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Current Research and Scholarly Interests

Expansion of gene regions containing nucleotide repeats (NRs) has a causal role in a variety of inherited degenerative neurological diseases, including Huntington’s Disease, certain spinocerebellar ataxias and muscular dystrophies, and some types of amyotrophic lateral sclerosis and frontotemporal dementia. A major area of investigation in our lab is study of mechanisms that selectively enable transcription through expanded NR regions in human genes. We also study the actions of abnormal mRNAs and proteins generated by such repeats, and efforts in the lab are aimed at treating these diseases by targeting expression of the abnormal genes. We also investigate mechanisms that underlie the occurrence of NR expansions.

Certain of our investigations are aimed at elucidating the signals that govern RNA decay. We use E. coli and Streptomyces species to investigate the mechanism of action of specific ribonucleases, related proteins, small non-coding RNAs, and to identify cellular events and proteins that regulate the actions of these molecules.

Our lab has long been interested in the evolution and dissemination of antibiotic resistance, and currently, we continue to pursue these interests by investigating the biology underlying the ability of bacteria to adapt non-mutationally to antibiotic exposure and other environmental stresses.

Abstract

Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

Abstract

Resistance of Staphylococcus aureus to beta-lactam antibiotics has led to increasing use of the glycopeptide antibiotic vancomycin as a life-saving treatment for major S. aureus infections. Coinfection by an unrelated bacterial species may necessitate concurrent treatment with a second antibiotic that targets the coinfecting pathogen. While investigating factors that affect bacterial antibiotic sensitivity, we discovered that susceptibility of S. aureus to vancomycin is reduced by concurrent exposure to colistin, a cationic peptide antimicrobial employed to treat infections by Gram-negative pathogens. We show that colistin-induced vancomycin tolerance persists only as long as the inducer is present and is accompanied by gene expression changes similar to those resulting from mutations that produce stably inherited reduction of vancomycin sensitivity (vancomycin-intermediate S. aureus [VISA] strains). As colistin-induced vancomycin tolerance is reversible, it may not be detected by routine sensitivity testing and may be responsible for treatment failure at vancomycin doses expected to be clinically effective based on such routine testing.Commonly, antibiotic resistance is associated with permanent genetic changes, such as point mutations or acquisition of resistance genes. We show that phenotypic resistance can arise where changes in gene expression result in tolerance to an antibiotic without any accompanying genetic changes. Specifically, methicillin-resistant Staphylococcus aureus (MRSA) behaves like vancomycin-intermediate S. aureus (VISA) upon exposure to colistin, which is currently used against infections by Gram-negative bacteria. Vancomycin is a last-resort drug for treatment of serious S. aureus infections, and VISA is associated with poor clinical prognosis. Phenotypic and reversible resistance will not be revealed by standard susceptibility testing and may underlie treatment failure.

Abstract

A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens.

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis.Proceedings of the National Academy of Sciences of the United States of AmericaJeong, S., Martchenko, M., Cohen, S. N.2013; 110 (42): E4007-15

Abstract

The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and β1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosisPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAJeong, S., Martchenko, M., Cohen, S. N.2013; 110 (42): E4007-E4015

DNA cloning: A personal view after 40 yearsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N.2013; 110 (39): 15521-15529

Abstract

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer's laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning.

Abstract

Pseudomembranous enterocolitis associated with Clostridium difficile infection is an important cause of morbidity and mortality in patients being treated with antibiotics. Two closely related large protein toxins produced by C. difficile, TcdA and TcdB, which act identically but at different efficiencies to glucosylate low-molecular-weight Rho GTPases, underlie the microbe's pathogenicity. Using antisense RNA encoded by a library of human expressed sequence tags (ESTs), we randomly inactivated host chromosomal genes in HeLa cells and isolated clones that survived exposure to ordinarily lethal doses of TcdB. This phenotypic screening and subsequent analysis identified solute carrier family 11 member 1 (SLC11A1; formerly NRAMP1), a divalent cation transporter crucial to host defense against certain microbes, as an enhancer of TcdB lethality. Whereas SLC11A1 normally is poorly expressed in human cells of nonmyeloid lineage, TcdB increased SLC11A1 mRNA abundance in such cells through the actions of the RNA-binding protein HuR. We show that short hairpin RNA (shRNA) directed against SLC11A1 reduced TcdB glucosylation of small Rho GTPases and, consequently, toxin lethality. Consistent with the previously known role of SLC11A1 in cation transport, these effects were enhanced by elevation of Mn(2+) in media; conversely, they were decreased by treatment with a chelator of divalent cations. Our findings reveal an unsuspected role for SLC11A1 in determining C. difficile pathogenicity, demonstrate the novel ability of a bacterial toxin to increase its cytotoxicity, establish a mechanistic basis for these effects, and suggest a therapeutic approach to mitigate cell killing by C. difficile toxins A and B.

Abstract

Escherichia coli cells normally require RNase E activity to form colonies (colony-forming ability [CFA]). The CFA-defective phenotype of cells lacking RNase E is partly reversed by overexpression of the related endoribonuclease RNase G or by mutation of the gene encoding the RNA helicase DeaD. We found that the carbon source utilization by rne deaD doubly mutant bacteria differs from that of rne(+) cells and from that of cells mutated in deaD alone and that the loss of rne function in these bacteria limits conversion of the glycolytic pathway product phosphoenolpyruvate to the tricarboxylic acid (TCA) cycle intermediate oxaloacetic acid. We show that the mechanism underlying this effect is reduced production of the enzyme phosphoenolpyruvate carboxylase (PPC) and that adventitious overexpression of PPC, which facilitates phosphoenolpyruvate utilization and connects the glycolytic pathway with the TCA cycle, restored CFA to rne deaD mutant bacteria cultured on carbon sources that otherwise were unable to sustain growth. We further show that bacteria producing full-length RNase E, which allows formation of degradosomes, have nutritional requirements different from those of cells supplied with only the N-terminal catalytic region of RNase E and that mitigation of RNase E deficiency by overexpression of a related RNase, RNase G, is also affected by carbon source. Our results reveal previously unsuspected effects of RNase E deficiency and degradosome formation on nutrient utilization by E. coli cells.

Abstract

Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC).PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways.Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup.The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.

Abstract

Plants, animals, bacteria, and Archaea all have evolved mechanisms to cope with environmental or cellular stress. Bacterial cells respond to the stress of DNA damage by activation of the SOS response, the canonical RecA/LexA-dependent signal transduction pathway that transcriptionally derepresses a multiplicity of genes-leading to transient arrest of cell division and initiation of DNA repair. Here we report the previously unsuspected role of E. coli endoribonuclease RNase E in regulation of the SOS response. We show that RNase E deletion or inactivation of temperature-sensitive RNase E protein precludes normal initiation of SOS. The ability of RNase E to regulate SOS is dynamic, as down regulation of RNase E following DNA damage by mitomycin C resulted in SOS termination and restoration of RNase E function leads to resumption of a previously aborted response. Overexpression of the RraA protein, which binds to the C-terminal region of RNase E and modulates the actions of degradosomes, recapitulated the effects of RNase E deficiency. Possible mechanisms for RNase E effects on SOS are discussed.

Abstract

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 proteinPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANabhan, J. F., Hu, R., Oh, R. S., Cohen, S. N., Lu, Q.2012; 109 (11): 4146-4151

Abstract

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.

Abstract

The outcome of exposure to infectious microbes or their toxins is influenced by both microbial and host genes. Some host genes encode defense mechanisms, whereas others assist pathogen functions. Genomic analyses have associated host gene mutations with altered infectious disease susceptibility, but evidence for causality is limited. Here we demonstrate that human genetic variation affecting capillary morphogenesis gene 2 (CMG2), which encodes a host membrane protein exploited by anthrax toxin as a principal receptor, dramatically alters toxin sensitivity. Lymphoblastoid cells derived from a HapMap Project cohort of 234 persons of African, European, or Asian ancestry differed in sensitivity mediated by the protective antigen (PA) moiety of anthrax toxin by more than four orders of magnitude, with 99% of the cohort showing a 250-fold range of sensitivity. We find that relative sensitivity is an inherited trait that correlates strongly with CMG2 mRNA abundance in cells of each ethnic/geographical group and in the combined population pool (P = 4 × 10(-11)). The extent of CMG2 expression in transfected murine macrophages and human lymphoblastoid cells affected anthrax toxin binding, internalization, and sensitivity. A CMG2 single-nucleotide polymorphism (SNP) occurring frequently in African and European populations independently altered toxin uptake, but was not statistically associated with altered sensitivity in HapMap cell populations. Our results reveal extensive human diversity in cell lethality dependent on PA-mediated toxin binding and uptake, and identify individual differences in CMG2 expression level as a determinant of this diversity. Testing of genomically characterized human cell populations may offer a broadly useful strategy for elucidating effects of genetic variation on infectious disease susceptibility.

Abstract

Host factors that microbial pathogens exploit for their propagation are potential targets for therapeuic countermeasures. No host enzyme has been identified whose genetic absence benefits the intact mammalian host in vivo during infection with Mycobacterium tuberculosis (Mtb), the leading cause of death from bacterial infection. Here, we report that the dsRNA-dependent protein kinase (PKR) is such an enzyme. PKR-deficient mice contained fewer viable Mtb and showed less pulmonary pathology than wild type mice. We identified two potential mechanisms for the protective effect of PKR deficiency: increased apoptosis of macrophages in response to Mtb and enhanced activation of macrophages in response to IFN-gamma. The restraining effect of PKR on macrophage activation was explained by its mediation of a previously unrecognized ability of IFN-gamma to induce low levels of the macrophage deactivating factor interleukin 10 (IL10). These observations suggest that PKR inhibitors may prove useful as an adjunctive treatment for tuberculosis.

Abstract

Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5'-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. However, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart: two substrate-bound tryptic peptides, (65) HGFLPLK (71)--which includes amino acids previously implicated in substrate binding and catalysis--and (24) LYDLDIESPGHEQK (37)--which includes the Q36 locus-were identified in wild-type enzyme complexes. Only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.

Abstract

Hypoxia-inducible factor-1α (HIF-1α) is a principal regulator of angiogenesis and other cellular responses to hypoxic stress in both normal and tumor cells. To identify novel mechanisms that regulate expression of HIF-1α, we designed a genome-wide screen for expressed sequence tags (ESTs) that when transcribed in the antisense direction increase production of the HIF-1α target, vascular endothelial growth factor (VEGF), in human breast cancer cells. We discovered that heat shock factor (HSF) proteins 2 and 4-which previously have been implicated in the control of multiple genes that modulate cell growth and differentiation and protect against effects of environmental and cellular stresses-function together to maintain a steady state level of HIF-1α transcription and VEGF production in these cells. We show both HSFs bind to discontinuous heat shock element (HSE) sequences we identified in the HIF-1α promoter region and that downregulation of either HSF activates transcription of HIF-1α. We further demonstrate that HSF2 and HSF4 displace each other from HSF/HSE complexes in the HIF-1α promoter so that HIF-1α transcription is also activated by overexpression of either HSFs. These results argue that HSF2 and HSF4 regulate transcription of HIF-1α and that a critical balance between these HSF is required to maintain HIF-α expression in a repressed state. Our findings reveal a previously unsuspected role for HSFs in control of VEGF and other genes activated by canonical HIF-1α-mediated signaling.

Heterodimeric integrin complexes containing beta 1-integrin promote internalization and lethality of anthrax toxinPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMartchenko, M., Jeong, S., Cohen, S. N.2010; 107 (35): 15583-15588

Abstract

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin beta1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to alpha4beta1- and alpha5beta1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8--with purified alpha5beta1 complex in vitro. Monoclonal antibody directed against beta1-integrin or its alpha integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid--which interferes with CD44-mediated integrin activation--had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that beta1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.

Abstract

A tetR family transcriptional regulatory gene (SCO1712) was identified as a global antibiotic regulatory gene from a Streptomyces interspecies DNA microarray analysis. SCO1712 disruption in Streptomyces coelicolor not only upregulated antibiotic biosynthesis through pathway-specific regulators when a previously identified pleiotropic downregulatory wblA was expressed but also further stimulated antibiotic production in a wblA deletion mutant, implying that SCO1712 might encode a novel antibiotic downregulator.

Abstract

RNase III family enzymes, which are perhaps the most widely conserved of all ribonucleases, are known primarily for their role in the processing and maturation of small RNAs. The RNase III gene of Streptomyces coelicolor, which was discovered initially as a global regulator of antibiotic production in this developmentally complex bacterial species and named absB (antibiotic biosynthesis gene B), has subsequently also been found to modulate the cellular abundance of multiple messenger RNAs implicated in morphological differentiation. We report here that regulation of differentiation-related mRNAs by the S. coelicolor AbsB/RNase III enzyme occurs largely by ribonucleolytic cleavage of transcripts encoding the pleiotropic transcription factor, AdpA, and that AdpA and AbsB participate in a novel feedback-control loop that reciprocally regulates the cellular levels of both proteins. Our results reveal a previously unsuspected mechanism for global ribonuclease-mediated control of gene expression in streptomycetes.

Abstract

Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimer's disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.

Abstract

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.

Microbial Drug Resistance: An Old Problem in Need of New Solutions.Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg. (D.A. Relman, M.A. Hamburg, E.R. Choffnes, and A. Mack, eds). Washington, DC: National Academies PressCohen SN2009: 173-180

Abstract

The broad cellular actions of RNase III family enzymes include ribosomal RNA (rRNA) processing, mRNA decay, and the generation of noncoding microRNAs in both prokaryotes and eukaryotes. Here we report that YmdB, an evolutionarily conserved 18.8-kDa protein of Escherichia coli of previously unknown function, is a regulator of RNase III cleavages. We show that YmdB functions by interacting with a site in the RNase III catalytic region, that expression of YmdB is transcriptionally activated by both cold-shock stress and the entry of cells into stationary phase, and that this activation requires the sigma-factor-encoding gene, rpoS. We discovered that down-regulation of RNase III activity occurs during both stresses and is dependent on YmdB production during cold shock; in contrast, stationary-phase regulation was unperturbed in YmdB-null mutant bacteria, indicating the existence of additional, YmdB-independent, factors that dynamically regulate RNase III actions during normal cell growth. Our results reveal the previously unsuspected role of ribonuclease-binding proteins in the regulation of RNase III activity.

Abstract

The egress of HIV particles from virus-infected cells is accomplished by the recruitment of proteins that normally mediate host cell endocytic functions. This process requires interaction of the HIV Gag protein with the host protein TSG101 (tumor susceptibility gene 101). Here, we report the use of a bacterial reverse two-hybrid system to identify cyclic peptides that interfere with the Gag-TSG101 interaction and the finding that a five amino acid peptide discovered by this approach can disrupt the interaction and consequently inhibit HIV egress. The inhibiting molecule, which was selected from a cyclic peptide library containing approximately 3.2 x 10(6) members, differs in primary sequence from the interacting sites of either TSG101 or Gag. Addition of cyclic peptide tagged with an HIV Tat sequence, which previously has been shown to enhance protein translocation across plasma membranes, to cultured human cells inhibited the production of virus-like particles (VLPs) by these cells (IC(50) of 7 microM), and this inhibition occurred in the absence of adverse affects on normal endocytic functions mediated by TSG101. A mutant Gag protein not dependent on TSG101 for release was unaffected by the cyclic peptide. Our findings, which suggest that interference with the TSG101-Gag interaction by cyclic peptides may be of practical use in the treatment of HIV infections, identify a specific cyclic peptide that reduces VLP release by this mechanism; they also demonstrate that the efficiency of interference with protein-protein interactions by cyclic peptides can be enhanced by tagging the peptides with translocation-promoting sequences. Collectively our results support the notion that small molecule therapeutics that inhibit specific interactions between viral and host proteins may have general applicability in antiviral therapy.

Abstract

The Streptomyces coelicolor absB gene encodes an RNase III family endoribonuclease and is normally essential for antibiotic biosynthesis. Here we report that AbsB controls its own expression by sequentially and site specifically cleaving stem-loop segments of its polycistronic transcript. Our results demonstrate a ribonucleolytic regulatory role for AbsB in vivo.

Abstract

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNA(Asn) precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity-implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.

Abstract

The limitation of proliferative potential in human somatic cells imposed by replicative senescence has been proposed as a mechanism of tumor suppression. The E3 ubiquitin ligase Smurf2 is up-regulated during replicative senescence in response to telomere shortening, and induces senescence when expressed adventitiously in early passage or telomerase-immortalized human fibroblasts. To investigate the generality of Smurf2's control of cell proliferation, we have studied the effects of Smurf2 up-regulation on cell proliferation in early passage human mammary epithelial cells which normally do not show elevated expression of Smurf2 during senescence, and in 16 human cancer cell lines derived from both sarcomas and carcinomas. Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.

Abstract

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis.Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells.Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

Abstract

Using Streptomyces coelicolor microarrays to discover regulators of gene expression in other Streptomyces species, we identified wblA, a whiB-like gene encoding a putative transcription factor, as a down-regulator of doxorubicin biosynthesis in Streptomyces peucetius. Further analysis revealed that wblA functions pleiotropically to control antibiotic production and morphological differentiation in streptomycetes. Our results reveal a novel biological role for wblA and show the utility of interspecies microarray analysis for the investigation of streptomycete gene expression.

Abstract

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

Abstract

Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75(MDM2) is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75(MDM2) doublets and p90(MDM2), which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90(MDM2). The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90(MDM2) promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.

Abstract

Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

Abstract

The filamentous bacterium, Streptomyces coelicolor, undergoes a complex cycle of growth and development in which morphological differentiation coincides with the activation of the orphan response regulator RamR and the biosynthesis of a morphogenic peptide called SapB. SapB is a lantibiotic-like molecule derived from the product of the ramS gene that promotes formation of aerial hyphae by breaking the aqueous tension on the surface of the substrate mycelium. A ramR-disrupted mutant is delayed in aerial hyphae formation while constitutive overexpression of ramR accelerates aerial hyphae formation in the wild-type strain and restores SapB biosynthesis and aerial hyphae formation in all developmental mutants (bld) tested. Using DNA microarrays to globally identify S. coelicolor genes whose transcription was affected by ramR mutation or overexpression, we discovered a ramR-activated locus of contiguous cotranscribed developmental genes that modulate both aerial hyphae formation and sporulation. The genes of this cluster of ramR-activated genes (rag), which are chromosomally distant from previously known RamR-regulated genes, include: ragA (sco4075) and ragB (sco4074), which encode two subunits of an ABC transporter, ragK (sco4073), a putative histidine kinase, and ragR (sco4072), a ramR paralogue. Promoter mapping and protein-DNA binding experiments indicate that RamR activates ragABKR transcription directly, by binding to three sequence motifs in the ragABKR promoter region. A constructed ragABKR null mutant was able to synthesize SapB and erect aerial hyphae; however, these hyphae were unusually branched, reminiscent of substrate hyphae. Subsequent stages of differentiation, septation and sporogenesis were delayed. The role of ragABKR in aerial hyphae formation was shown both by epistasis (ragR-activated aerial hyphae formation in bld mutants) and extracellular complementation (ragR-induced synthesis of an activity allowing aerial hyphae formation in bld mutants) experiments. In conclusion, the ragABKR locus activates a SapB-independent developmental pathway that is involved in both aerial hyphae formation and sporulation, serving to integrate sequential morphogenic changes.

Abstract

African swine fever virus (ASFV) produces a fatal acute hemorrhagic fever in domesticated pigs that potentially is a worldwide economic threat. Using an expressed sequence tag (EST) library-based antisense method of random gene inactivation and a phenotypic screen for limitation of ASFV replication in cultured human cells, we identified six host genes whose cellular functions are required by ASFV. These included three loci, BAT3 (HLA-B-associated transcript 3), C1qTNF (C1q and tumor necrosis factor-related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis-related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Our results, which demonstrate the utility of EST-based functional screens for the detection of host genes exploited by pathogenic viruses, reveal a novel collection of cellular genes previously not known to be required for ASFV infection.

Abstract

Using transcripts initiated at a chromosomally integrated retrovirus-based promoter to perturb gene expression randomly in human prostate cancer cells, we isolated cell clones resistant to taxane lethality and discovered the role of a previously uncharacterized gene, txr1, in this phenotype. We show that txr1 impedes taxane-induced apoptosis in tumor cells by transcriptionally down-regulating the production of thrombospondin-1 (TSP-1)--known earlier for both its anti-angiogenic and proapoptotic actions. Decrease of Txr1 or treatment with TSP-1 or TSP-1 mimetic peptide sensitized cells to taxane cytotoxicity by activating signaling through the CD47 receptor (also known as the integrin-associated protein), whereas interference with CD47 function reduced taxane-induced cell death. Cellular abundance of Txr1 and TSP-1 varied inversely, and alteration of the level of both proteins correlated highly with taxol resistance in 13 of 19 NCI-60 cancer cell lines. Our results reveal a hitherto unsuspected mechanism of taxane resistance, elucidate the role of txr1 in this resistance, and identify txr1 as a regulator of TSP-1 production and an agent for its chemotherapeutic modulation.

Abstract

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor-specific changes in degradosome composition--and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

Abstract

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Abstract

Toxins produced by Bacillus anthracis and other microbial pathogens require functions of host cell genes to yield toxic effects. Here we show that low density lipoprotein receptor-related protein 6 (LRP6), previously known to be a coreceptor for the Wnt signaling pathway, is required for anthrax toxin lethality in mammalian cells. Downregulation of LRP6 or coexpression of a truncated LRP6 dominant-negative peptide inhibited cellular uptake of complexes containing the protective antigen (PA) carrier of anthrax toxin moieties and protected targeted cells from death, as did antibodies against epitopes in the LRP6 extracellular domain. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with PA receptors TEM8/ATR and/or CMG2 to form a multicomponent complex that enters cells upon PA binding. Our results, which reveal a previously unsuspected biological role for LRP6, identify LRP6 as a potential target for countermeasures against anthrax toxin lethality.

Abstract

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.

Effects of threshold choice on biological conclusions reached during analysis of gene expression by DNA microarraysPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAPan, K. H., Lih, C. J., Cohen, S. N.2005; 102 (25): 8961-8965

Abstract

Global analysis of gene expression by using DNA microarrays is employed increasingly to search for differences in biological properties between normal and diseased tissue. In such studies, expression that deviates from defined thresholds commonly is used for creating genetic signatures that characterize disease vs. normality. Although it is axiomatic that the threshold parameters applied to microarray analysis will alter the contents of such genetic signatures, the extent to which threshold choice can affect the fundamental conclusions made from microarray-based studies has not been elucidated. We used GABRIEL (Genetic Analysis By Rules Incorporating Expert Logic), a platform of knowledge-based algorithms for the global analysis of gene expression, together with conventional statistical approaches, to examine the sensitivity of conclusions to threshold choice in recently published microarray-based studies. An analysis of the effects of threshold decisions in one of these studies [Ramaswamy, S., Ross, K. N., Lander, E. S. & Golub, T. R. (2003) Nat. Genet. 33, 49-54], which arrived at the important conclusion that the metastatic potential of primary tumors is encoded by the bulk of cells in the tumor, is the focus of this article. We discovered that support for this conclusion highly depends on the threshold used to create gene expression signatures. We also found that threshold choice dramatically affected the gene function categories represented nonrandomly in signatures. Our results suggest that the robustness of biological conclusions made by using microarray analysis should be routinely assessed by examining the validity of the conclusions by using a range of threshold parameters.

Abstract

Based on the chromosomal locations of genes inferred from sequence analysis to be essential for the viability of Streptomyces coelicolor, Bentley et al. [Bentley, S.D., et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2), Nature 417, 141-147.] have suggested that a 4.9 Mb central region of the linear S. coelicolor chromosome encodes 'core' functions expressed during vegetative growth of this species, while 1.5 Mb and 2.3 Mb chromosomal DNA segments lateral to this core encode auxiliary functions proposed to be required under other growth conditions. To examine this hypothesis and experimentally identify genes expressed during vegetative growth of S. coelicolor cultures, we used DNA microarrays to measure globally the abundance of S. coelicolor transcripts in cells growing in liquid medium. We found that, overall, genes corresponding to the 4.9 Mb core region of the S. coelicolor M145 chromosome were more highly expressed under non-limiting growth conditions than genes in the 1.5 Mb left and 2.3 Mb right chromosome arms, supporting the notion of the core versus auxiliary organization of genes on the chromosome. To examine how this chromosomal distribution of transcripts changes under other growth conditions, we also measured gene expression changes during stationary phase and several stress conditions. During stationary phase, the composition of S. coelicolor transcripts appears to shift from large quantities of growth-related transcripts encoded in the core region to those of less characterized genes, which may be essential for differentiation and other physiological responses, encoded throughout the chromosome. After temperature and osmotic upshifts, we found that S. coelicolor transiently induces a set of several hundred genes located throughout the chromosome, which may function in response mechanisms common to the two stress conditions.

Abstract

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT-PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.

Abstract

Progressive telomere shortening activates replicative senescence, which prevents somatic cells from being propagated indefinitely in culture. The limitation of proliferative capacity imposed by replicative senescence is thought to contribute to both organismal aging and the prevention of tumor development. Here we report that up-regulation of Smurf2, an E3 ubiquitin ligase previously implicated in TGF-beta signaling, is a specific consequence of telomere attrition in human fibroblasts and that such up-regulation is sufficient to produce the senescence phenotype. Adventitious production of the Smurf2 protein in early passage fibroblasts at the same physiological level observed during telomere-mediated senescence resulted in proliferative arrest in a viable state, morphological and biochemical alterations characteristic of senescence, acquisition of senescence-specific alterations in gene expression, and reversal of cellular immortalization by telomerase. We show that the senescence-inducing actions of Smurf2 occur in the absence of detectable DNA damage or stress response, that Smurf2's effects require a novel function distinct from its E3 activity, that Smurf2 recruits the Rb and p53 pathways for senescence induction, and that while p21 is elevated by Smurf2, Smurf2-mediated senescence is independent of p21. Smurf2 is the first gene found to be both up-regulated by telomere attrition and sufficient to induce senescence.

Abstract

The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety. During B. anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol. To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of approximately 40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death. This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity. ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity. Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.

Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complexPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABao, K., Cohen, S. N.2004; 101 (40): 14361-14366

Abstract

Replication of Streptomyces linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5' termini that are extended by a telomere binding complex. This complex contains both a telomere-protecting terminal protein (Tpg) and a telomere-associated protein that interacts with Tpg and the DNA ends of linear Streptomyces replicons. By using histidine-tagged telomere-associated protein (Tap) as a scaffold, we identified DNA polymerase (PolA) and topoisomerase I (TopA) proteins as other components of the Streptomyces telomere complex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase (RT) activity in addition to their predicted functions. Although RT activity innate to other DNA-dependent DNA polymerases has been observed previously, its occurrence in a topoisomerase is unprecedented. Deletion mapping and sequence analysis showed that the RT activity of Streptomcyces TopA resides in a peptide region containing motifs that are absent from most bacterial topoisomerases but are highly conserved in a novel subfamily of eubacterial topoisomerases found largely in Actinobacteria. Within one of these motifs, and essential to the RT function of Streptomyces TopA, is an Asp-Asp doublet sequence required also for the RT activities of human immunodeficiency virus and eukaryotic cell telomerases.

Abstract

Telomere shortening in populations of human mammary epithelial cells (HMECs) that survive early replicative arrest (M0) by the inactivation of p16(INK4A) during cell culture on plastic dishes leads to a state of permanent replicative arrest termed senescence. While culture of HMECs on feeder layers abrogates M0 and p16(INK4A) inactivation, progressive telomere attrition in these cells also eventually results in permanent replicative arrest. Expression of telomerase prevents both senescence on plastic (S-P) and senescence on feeder layers (S-FL) in HMECs, as it does also in cultured primary human fibroblasts. We report here that the gene expression profiles of senescence in HMECs of the same lineage maintained under different culture conditions showed surprisingly little commonality. Moreover, neither of these senescence-associated profiles in HMECs resembles the profile for senescence in human fibroblasts. These results indicate that senescence-associated alterations in gene expression resulting from telomere attrition are affected by culture conditions as well as by cell origins, and argue that replicative senescence at the molecular level is a diverse rather than unique cellular process.

Global analysis of Escherichia coli RNA degradosome function using DNA microarraysPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABernstein, J. A., Lin, P. H., Cohen, S. N., Lin-Chao, S.2004; 101 (9): 2758-2763

Abstract

RNase E, an essential endoribonuclease of Escherichia coli, interacts through its C-terminal region with multiple other proteins to form a complex termed the RNA degradosome. To investigate the degradosome's proposed role as an RNA decay machine, we used DNA microarrays to globally assess alterations in the steady-state abundance and decay of 4,289 E. coli mRNAs at single-gene resolution in bacteria carrying mutations in the degradosome constituents RNase E, polynucleotide phosphorylase, RhlB helicase, and enolase. Our results show that the functions of all four of these proteins are necessary for normal mRNA turnover. We identified specific transcripts and functionally distinguishable transcript classes whose half-life and abundance were affected congruently by multiple degradosome proteins, affected differentially by mutations in degradosome constituents, or not detectably altered by degradosome mutations. Our results, which argue that decay of some E. coli mRNAs in vivo depends on the action of assembled degradosomes, whereas others are acted on by degradosome proteins functioning independently of the complex, imply the existence of structural features or biochemical factors that target specific classes of mRNAs for decay by degradosomes.

Abstract

The chromosomes of several widely used laboratory derivatives of Streptomyces coelicolor A3(2) were found to have 1.06 Mb inverted repeat sequences at their termini (i.e. long-terminal inverted repeats; L-TIRs), which are 50 times the length of the 22 kb TIRs of the sequenced S. coelicolor strain M145. The L-TIRs include 1005 annotated genes and increase the overall chromosome size to 9.7 Mb. The 1.06 Mb L-TIRs are the longest reported thus far for an actinomycete, and are proposed to represent the chromosomal state of the original soil isolate of S. coelicolor A3(2). S. coelicolor A3(2), M600 and J1501 possess L-TIRs, whereas approximately half the examined early mutants of A3(2) generated by ultraviolet (UV) or X-ray mutagenesis have truncated their TIRs to the 22 kb length. UV radiation was found to stimulate L-TIR truncation. Two copies of a transposase gene (SCO0020) flank 1.04 Mb of DNA in the right L-TIR, and recombination between them appears to generate strains containing short TIRs. This TIR reduction mechanism may represent a general strategy by which transposable elements can modulate the structure of chromosome ends. The presence of L-TIRs in certain S. coelicolor strains represents a major chromosomal alteration in strains previously thought to be genetically similar.

Abstract

A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40:1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor polynucleotide phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.

Abstract

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.

Abstract

The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size-both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB-both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids-reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

Abstract

Ribonuclease E (RNase E) has a key role in mRNA degradation and the processing of catalytic and structural RNAs in E. coli. We report the discovery of an evolutionarily conserved 17.4 kDa protein, here named RraA (regulator of ribonuclease activity A) that binds to RNase E and inhibits RNase E endonucleolytic cleavages without altering cleavage site specificity or interacting detectably with substrate RNAs. Overexpression of RraA circumvents the effects of an autoregulatory mechanism that normally maintains the RNase E cellular level within a narrow range, resulting in the genome-wide accumulation of RNase E-targeted transcripts. While not required for RraA action, the C-terminal RNase E region that serves as a scaffold for formation of a multiprotein degradosome complex modulates the inhibition of RNase E catalytic activity by RraA. Our results reveal a possible mechanism for the dynamic regulation of RNA decay and processing by inhibitory RNase binding proteins.

Abstract

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.

Abstract

The filamentous bacterium Streptomyces coelicolor differentiates by forming specialized, spore-bearing aerial hyphae that grow into the air. Using microarrays, we identified genes that are down-regulated in a mutant unable to erect aerial hyphae. Through this route, we identified a previously unknown layer of aerial mycelium surface proteins (the "chaplins"). The chaplins share a hydrophobic domain of approximately 40 residues (the "chaplin domain"), and all have a secretion signal. The five short chaplins (ChpD,E,F,G,H) have one chaplin domain, whereas the three long chaplins (ChpA,B,C) have two chaplin domains and a C-terminal "sorting signal" that targets them for covalent attachment to the cell wall by sortase enzyme. Expression of the two chaplin genes examined (chpE, chpH) depended on aerial hyphae formation but not sporulation, and egfp fusions showed their expression localized to aerial structures. Mass spectrometry of cell wall extracts confirmed that the short chaplins localized to the cell surface. Deletion of chaplin genes caused severe delays in aerial hyphae formation, a phenotype rescued by exogenous application of chaplin proteins. These observations implicate the chaplins in aerial mycelium formation, and suggest that coating of the envelope by the chaplins is required for aerial hyphae to grow out of the aqueous environment of the substrate mycelium into the air.

TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALu, Q., Hope, L. W., Brasch, M., Reinhard, C., Cohen, S. N.2003; 100 (13): 7626-7631

Abstract

Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (tsg101) recently was found to be centrally involved in this process. Here we report that TSG101 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), an early endosomal protein, and that disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevented delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. Our results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes.

Abstract

Replicative senescence is the state of irreversible proliferative arrest that occurs as a concomitant of progressive telomere shortening. By using cDNA microarrays and the gabriel system of computer programs to apply domain-specific and procedural knowledge for data analysis, we investigated global changes in gene transcription occurring during replicative senescence in human fibroblasts and mammary epithelial cells (HMECs). Here we report the identification of transcriptional "fingerprints" unique to senescence, the finding that gene expression perturbations during senescence differ greatly in fibroblasts and HMECs, and the discovery that despite the disparate nature of the chromosomal loci affected by senescence in fibroblasts and HMECs, the up-regulated loci in both types of cells show physical clustering. This clustering, which contrasts with the random distribution of genes down-regulated during senescence or up-regulated during reversible proliferative arrest (i.e., quiescence), supports the view that replicative senescence is associated with alteration of chromatin structure.

Abstract

Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates. Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons. We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process. Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini. Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA. Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs. Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends. Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates. They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.

The catalytic domain of RNase E shows inherent 3 ' to 5 ' directionality in cleavage site selectionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAFeng, Y. A., Vickers, T. A., Cohen, S. N.2002; 99 (23): 14746-14751

Abstract

RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2'-O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5'-monophosphate RNA termini but has an inherent mode of cleavage in the 3' to 5' direction. Target sequences made uncleavable by the introduction of 2'-O-methyl-modified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5' to, but not 3' to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3' to 5' "scanning" mechanism and imply that anchoring of the enzyme to the 5'-monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.

Abstract

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non-homologous end-to-end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non-recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end-to-end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.

Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarraysPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABernstein, J. A., Khodursky, A. B., Lin, P. H., Lin-Chao, S., Cohen, S. N.2002; 99 (15): 9697-9702

Abstract

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.

Abstract

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.

Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1)PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAOh, H., Mammucari, C., Nenci, A., Cabodi, S., Cohen, S. N., Dotto, G. P.2002; 99 (8): 5430-5435

Abstract

TSG101 was discovered in a screen for tumor susceptibility genes and has since been shown to have a multiplicity of biological effects. However, the basis for TSG101's ability to regulate cell growth has not been elucidated. We report here that the TSG101 protein binds to the cyclin/cyclin-dependent kinase (CDK) inhibitor (CKI) p21(Cip1/WAF1) and increases stability of the p21 protein in HEK293F cells and differentiating primary keratinocytes, suppressing differentiation in a p21-dependent manner. In proliferating keratinocytes where the p21 protein is relatively stable, TSG101 does not affect the stability or expression of p21 but shows p21-dependent recruitment to cyclin/CDK complexes, inhibits cyclin/CDK activity, and causes strong growth suppression to a much greater extent in p21+/+ than in p21-/- cells. Conversely, suppression of endogenous TSG101 expression by an antisense TSG101 cDNA causes doubling of the fraction of keratinocytes in the S phase of the cell cycle as occurs during p21 deficiency. Our results indicate that TSG101 has a direct role in the control of growth and differentiation in primary epithelial cells, and that p21 is an important mediator of these TSG101 functions.

Abstract

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules.

Abstract

Growth arrest-specific (Gas) genes are expressed during serum starvation or contact inhibition of cells grown in culture. Here we report the isolation and characterization of Gas8, a novel gene identified on the basis of its growth arrest-specific expression in murine fibroblasts. We show that production of Gas8 mRNA and protein occurs in adult mice predominantly in the testes, where expression is regulated during postmeiotic development of male gametocytes. Whereas a low level of Gas8 mRNA was detected by Northern blotting in testes of murine male neonates and young adolescents, Gas8 mRNA increased rapidly postmeiotically. In adult males, both Gas8 mRNA and protein reached steady state levels in testes that were 10-fold higher than in other tissues. Immunohistochemical analyses showed that Gas8 protein accumulates in gametocytes as they approach the lumen of seminiferous tubules and is localized to the cytoplasm of round spermatids, the tails of elongating spermatids, and mature spermatid tail bundles protruding into the lumen; in epididymal spermatozoa Gas8 protein was present in the flagella. However, premeiotic murine gametocytes lacked detectable Gas8 protein, as did seminiferous tubules in biopsy specimens from seven human males having cytological evidence of non-obstructive azoospermia secondary to Sertoli cell-only syndrome. Our findings, which associate Gas8 production developmentally with the later stages of spermatogenesis and spatially with the sperm motility apparatus, collectively suggest that this growth arrest-specific gene product may have a role in sperm motility. This postulated role for Gas8 is supported by our observation that highly localized production of Gas8 protein occurs also in the cilia of epithelial cells lining pulmonary bronchi and fallopian tubes and by the flagellar association of a Trypanosoma brucei ortholog of Gas8.

Analysis of DNA microarrays using algorithms that employ rule-based expert knowledgePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAPan, K. H., Lih, C. J., Cohen, S. N.2002; 99 (4): 2118-2123

Abstract

The ability to investigate the transcription of thousands of genes concurrently by using DNA microarrays offers both major scientific opportunities and significant analytical challenges. Here we describe GABRIEL, a rule-based system of computer programs designed to apply domain-specific and procedural knowledge systematically and uniformly for the analysis and interpretation of data from DNA microarrays. GABRIEL'S problem-solving rules direct stereotypical tasks, whereas domain-specific knowledge pertains to gene functions and relationships or to experimental conditions. Additionally, GABRIEL can learn novel rules through genetic algorithms, which define patterns that best match the data being analyzed and can identify groupings in gene expression profiles preordered by chromosomal position or by a nonsupervised algorithm such as hierarchical clustering. GABRIEL subsystems explain the logic that underlies conclusions and provide a graphical interface and interactive platform for the acquisition of new knowledge. The present report compares GABRIEL'S output with published findings in which expert knowledge has been applied post hoc to microarray groupings generated by hierarchical clustering.

Abstract

The eubacterial species Streptomyces coelicolor proceeds through a complex growth cycle in which morphological differentiation/development is associated with a transition from primary to secondary metabolism and the production of antibiotics. We used DNA microarrays and mutational analysis to investigate the expression of individual genes and multigene antibiotic biosynthetic pathways during these events. We identified expression patterns in biosynthetic, regulatory, and ribosomal protein genes that were associated highly specifically with particular stages of development. A knowledge-based algorithm that correlates temporal changes in expression with chromosomal position identified groups of contiguous genes expressed at discrete stages of morphological development, inferred the boundaries of known antibiotic synthesis gene loci, and revealed novel physical clusters of coordinately regulated genes. Microarray analysis of RNA from cells mutated in genes regulating synthesis of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) identified proximate and distant sites that contain putative ABC transporter and two-component system genes expressed coordinately with genes of specific biosynthetic pathways and indicated the existence of two functionally and physically discrete regulons in the Red pathway.

Abstract

Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.

Abstract

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.

Abstract

The pcnB gene, which encodes the principal poly(A) polymerase of Escherichia coli, promotes 3'-polyadenylation and chemical decay of mRNA. However, there is no evidence that pcnB-mediated mRNA destabilization decreases protein synthesis, suggesting that polyadenylation may enhance translational efficiency. Using in vitro translation by E. coli cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase (CAT) and beta-galactosidase genes to investigate this notion, we found no effect of poly(A) tails on protein synthesis. However, we observed that 3'-polyguanylation delayed the chemical decay of CAT mRNA and, even more dramatically, increased the ability of CAT mRNA to produce enzymatically active full-length protein in 30 S E. coli cell fractions. This resulted from interference with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather than endonucleolytic fragmentation by RNase E. Using bacteriophage T7 RNA polymerase to install poly(G) tails on mRNAs transcribed from polymerase chain reaction-generated DNA templates, we observed sharply increased synthesis of active proteins in vitro in coupled transcription/translation reactions. The ability of poly(G) tails to functionally stabilize transcripts from polymerase chain reaction-generated templates allows proteins encoded by translational open reading frames on genomic DNA or cDNA to be synthesized directly and efficiently in vitro.

Abstract

Linear plasmids and chromosomes of the bacterial genus Streptomyces have proteins of unknown characteristics and function linked covalently to their 5' DNA termini. We purified protein attached to the end of the pSLA2 linear plasmid of Streptomyces rochei, determined the N-terminal amino acid sequence, and used this information to clone corresponding genes from a S. rochei cosmid library. Three separate terminal protein genes (here designated as tpgR1, tpgR2, and tpgR3), which map to the S. rochei chromosome and to 100-kb and 206-kb linear plasmids contained in S. rochei, were isolated and found to encode a family of similar but distinct 21-kD proteins. Using tpgR1 to probe a genomic DNA library of Streptomyces lividans ZX7, whose linear chromosome can undergo transition to a circular form, we isolated a S. lividans chromosomal gene (tpgL) that we found specifies a protein closely related to, and functionally interchangeable with, TpgR proteins for pSLA2 maintenance in S. lividans. Mutation of tpgL precluded propagation of the pSLA2 plasmid in a linear form and also prevented propagation of S. lividans cells that contain linear, but not circular, chromosomes, indicating a specific and essential role for tpg genes in linear DNA replication. Surprisingly, Tpg proteins were observed to contain a reverse transcriptase-like domain rather than sequences in common with proteins that attach covalently to the termini of linear DNA replicons.

Abstract

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin (ACT) is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor-generating the deep-blue colonies responsible for the S. coelicolor name. However, multiple copies of the two regulatory genes, afsR and afsR2, activate ACT production in S. lividans, indicating that this streptomycete encodes a functional ACT biosynthetic pathway. Here we report that the occurrence of ACT biosynthesis in S. lividans is determined conditionally by the carbon source used for culture. We found that the growth of S. lividans on solid media containing glucose prevents ACT production in this species by repressing the synthesis of afsR2 mRNA; a shift to glycerol as the sole carbon source dramatically relieved this repression, leading to extensive ACT synthesis and obliterating this phenotypic distinction between S. lividans and S. coelicolor. Transcription from the afsR2 promoter during growth in glycerol was dependent on afsR gene function and was developmentally regulated, occurring specifically at the time of aerial mycelium formation and coinciding temporally with the onset of ACT production. In liquid media, where morphological differentiation does not occur, ACT production in the absence of glucose increased as S. lividans cells entered stationary phase, but unlike ACT biosynthesis on solid media, occurred by a mechanism that did not require either afsR or afsR2. Our results identify parallel medium-dependent pathways that regulate ACT biosynthesis in S. lividans and further demonstrate that the production of this antibiotic in S. lividans grown on agar can be modulated by carbon source through the regulation of afsR2 mRNA synthesis.

Abstract

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse-chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

Abstract

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101-/- embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

RNA degradosomes exist in vivo in Escherichia coli as multicomponent complexes associated with the cytoplasmic membrane via the N-terminal region of ribonuclease EPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALiou, G. G., Jane, W. N., Cohen, S. N., Lin, N. S., Lin-Chao, S.2001; 98 (1): 63-68

Abstract

RNase E isolated from Escherichia coli is contained in a multicomponent "degradosome" complex with other proteins implicated in RNA decay. Earlier work has shown that the C-terminal region of RNase E is a scaffold for the binding of degradosome components and has identified specific RNase E segments necessary for its interaction with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase. Here, we report electron microscopy studies that use immunogold labeling and freeze-fracture methods to show that degradosomes exist in vivo in E. coli as multicomponent structures that associate with the cytoplasmic membrane via the N-terminal region of RNase E. Whereas PNPase and enolase are present in E. coli in large excess relative to RNase E and therefore are detected in cells largely as molecules unlinked to the RNase E scaffold, immunogold labeling and biochemical analyses show that helicase is present in approximately equimolar amounts to RNase E at all cell growth stages. Our findings, which establish the existence and cellular location of RNase E-based degradosomes in vivo in E. coli, also suggest that RNA processing and decay may occur at specific sites within cells.

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGYPettis, G. S., Cohen, S. N.2001; 79 (3-4): 247-250

Abstract

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.

Abstract

Sam68 is a multimeric 68-kDa RNA-binding nuclear protein of unknown function that interacts with, and is tyrosine-phosphorylated by, the oncogenic protein Src during mitosis. Random homozygous knock-out (RHKO) is a retroviral-based antisense RNA strategy that can identify chromosomal genes whose functional disablement leads to reversible tumorigenic capabilities. Here we report that RHKO-induced Sam68 deficiency results in neoplastic transformation of murine NIH3T3 fibroblasts. Whereas simple haploinsufficiency of Sam68 produced by insertion mutagenesis in a single chromosomal allele did not detectably affect cell growth, reduction of Sam68 protein to <25% of the wild type level was associated with anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice. These properties were reversed by cessation of RHKO inactivation. Our findings, which indicate that the Sam68 protein level can prominently affect cell proliferation, implicate Sam68 function in tumorigenesis. Consistent with these results is evidence that cells undergoing mitosis show a dramatic reduction in the level of Sam68 protein.

Abstract

We previously have described the use of an antisense RNA strategy termed random homozygous knock-out (RHKO) to identify negative regulators of cell proliferation. Here we report the discovery that RHKO-mediated deficiency of the nCL-4 calpain protease results in cellular transformation of and tumorigenesis by murine NIH3T3 fibroblasts. We isolated cell clones able to form colonies on 0.5% soft agar and found that these cells generated tumors when injected subcutaneously into nude mice. The gene inactivated by RHKO was identified as nCL-4 by genomic library screening, transcript analysis, and DNA sequencing. Anchorage-independent growth, as indicated by colony formation on soft agar, was reversed by reversal of antisense-mediated homozygous inactivation, but continued haplo-insufficiency of nCL-4 resulting from insertional mutagenesis of one nCL-4 allele was associated with persistent tumorigenesis. nCL-4 cDNA expressed in naive 3T3 cells in the antisense, but not sense, direction under control of the cytomegalovirus early promoter reproduced the anchorage-independent growth effects of RHKO. Our results implicate deficiency of the nCL-4 calpain protease in neoplastic transformation.

Abstract

The tra gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211-6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.

Abstract

Long inverted repeats (palindromes) are ubiquitous among prokaryotic and eukaryotic genomes. Earlier work has implicated both DNA breaks and short inverted repeats (IRs) in the formation of long palindromes in yeast and Tetrahymena by a proposed mechanism of intramolecular recombination. Here we report that long-palindromic linear plasmids are formed in Streptomyces following double strand DNA breakage by a nonrecombinational intra-strand annealing process that also involves IRs. By modification of palindrome-generating linear plasmids and development of a novel procedure that enables the sequencing of palindrome junctions, we show that long-palindrome formation occurs by unimolecular intra-strand annealing of IRs followed by 3' extension of the resulting DNA fold-back. The consequent hairpin structures serve as templates for synthesis of duplex linear plasmids containing long palindromes. We suggest that this model for long-palindrome formation in Streptomyces may represent a generally applicable mechanism for generating DNA palindromes.

Abstract

In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable. The selective susceptibility of unstable Escherichia coli RNAs to 3' polyadenylation by the pcnB gene product, poly(A) polymerase I (PAP I), in vivo is a key factor in their rapid degradation by 3' to 5' exonucleases. Using highly purified His-tagged recombinant PAP I, we show that differential adenylation of RNA substrates by PAP I occurs in vitro and that this capability resides in PAP I itself rather than in any ancillary protein(s). Surprisingly, the efficiency of 3' polyadenylation is affected by substrate structure at both termini; single-strand segments at either the 5' or 3' end of RNA molecules and monophosphorylation at an unpaired 5' terminus dramatically increase the rate and length of 3' poly(A) tail additions by PAP I. Our results provide a mechanistic basis for the susceptibility of certain RNAs to 3' polyadenylation. They also suggest a model of "programmed" RNA decay in which endonucleolytically generated RNA fragments containing single-stranded monophosphorylated 5' termini are targeted for poly(A) addition and further degradation.

Abstract

Antisense inactivation of the tsg101 tumor susceptibility gene in murine NIH3T3 fibroblasts leads to neoplastic transformation and tumorigenesis, which are reversed by restoration of tsg101 activity. tsg101 deficiency is associated with a series of mitosis-related abnormalities, whereas overexpression of TSG101 can also result in neoplastic transformation and the perturbation of cell cycling. Together, these observations imply that TSG101 production outside of a narrow range can lead to abnormal cell growth. We report here that the TSG101 protein is maintained at an almost constant steady-state level in cultured murine and human cells and that this occurs through a posttranslational process involving TSG101 protein degradation. Sustained overproduction of TSG101 from chromosomally inserted adventitious constructs resulted in compensatory down-regulation of endogenous TSG101 and replacement of the native protein by the adventitious one. Using deletion mutants of TSG101, we mapped the region responsible for autoregulation of the TSG101 steady-state level to an evolutionarily conserved sequence, here termed the "steadiness box," located near TSG101's COOH-terminal end. Our results suggest a model in which the biological effects of TSG101 are modulated either by self-promoted proteolysis or participation with other cellular protein(s) in a proteolytic feedback-control loop.

Abstract

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.

Abstract

ARD-1 is an endoribonuclease identified initially as the product of a human cDNA that complements mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine nuclear extracts as an inhibitor of protein phosphatase-1. Earlier work has shown that the protein-coding sequence of ARD-1 is identical to the carboxy-terminal third of NIPP-1. However, whether ARD-1 is present in eukaryotes as a distinct entity has been unclear, as neither ARD-1-specific transcripts nor ARD-1 protein were detected in mammalian cells in earlier studies. Here we show that ARD-1 exists in human cells as a discrete protein, and that the ARD-1 and NIPP-1 peptides are isoforms encoded by a single gene and the same alternatively spliced precursor RNA. A retained intron containing multiple translation stop codons that are configured to terminate translation and initiate nonsense-mediated decay, limits the production of cellular ARD-1 protein. Our results establish the process by which functionally disparate ARD-1 and NIPP-1 peptides are generated from the protein-coding sequence of the same gene in human cells.

Abstract

SLP1(int) is a 17.2-kb genetic element that normally is integrated site specifically into the chromosome of Streptomyces coelicolor A3(2). The imp operon within SLP1(int) represses replication of both chromosomally integrated and extrachromosomal SLP1. During mating with S. lividans, SLP1(int) can excise, delete part of imp, and form a family of autonomously replicating conjugative plasmids. Earlier work has shown that impA and impC gene products act in concert to control plasmid maintenance and regulate their own transcription. Here we report that these imp genes act also on a second promoter, P(opimp) (promoter opposite imp), located adjacent to, and initiating transcription divergent from, imp to regulate loci involved in the intramycelial transfer of SLP1 plasmids. spdB1 and spdB2, two overlapping genes immediately 3' to P(opimp) and directly regulated by imp, are shown by Tn5 mutagenesis to control transfer-associated growth inhibition (i.e., pocking). Additional genes resembling transfer genes of other Streptomyces spp. plasmids and required for SLP1 transfer and/or postconjugal intramycelial spread are located more distally to P(opimp). Expression of impA and impC in an otherwise competent recipient strain prevented SLP1-mediated gene transfer of chromosomal and plasmid genes but not plasmid-independent chromosome-mobilizing activity, suggesting that information transduced to recipients after the formation of mating pairs affects imp activity. Taken together with earlier evidence that the imp operon regulates SLP1 DNA replication, the results reported here implicate imp in the overall regulation of functions related to the extrachromosomal state of SLP1.

Abstract

Functional inactivation of the tsg101 gene in mouse fibroblasts leads to cell transformation and the ability to form metastatic tumors in nude mice. Abnormal TSG101 transcripts with highly-specific deletions in the protein-coding region have been identified in human tumor samples and cancer cell lines, including prostate and breast carcinomas, and have been attributed to alternative splicing of TSG101 mRNA. The function of the TSG101 protein is not known, although its predicted sequence has suggested that it may function as a transcription factor.Human TSG101 N-terminal (encoding amino acids 10-240) and C-terminal (encoding amino acids 230-391) fragments were cloned and used in both transient transfection and protein binding experiments. The transient transfections were carried in CV-1 cells. Protein-protein interactions were determined by both glutathione-S-transferase fusion protein binding and co-immunoprecipitation.The N-terminal region of TSG101, when fused to the GAL4 DNA binding domain, can activate transcription; whereas the C-terminal region mediates transcriptional repression. Full-length TSG101 or its separated regions repressed ligand-dependent transcriptional activation by nuclear receptors, including androgen receptor and estrogen receptor, which play central roles in prostate carcinoma and breast carcinoma, respectively. In addition, a direct association between TSG101 and the transcriptional co-factor p300 was demonstrated in vitro and in vivo.These results indicate that TSG101 can function as a transcription modulator to affect nuclear receptor-mediated transcriptional activation, which raises the possibility that the tumor suppression by TSG101 observed previously may be mediated at least in part by its effects on nuclear receptor function.

Abstract

Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilator-stimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.

Abstract

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that 'nuclear inhibitor of PP1' (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a 'dominant negative' mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

Abstract

Growth arrest-specific (gas) genes are expressed preferentially in cells that enter a quiescent state. gas7, which we identified in serum-starved murine fibroblasts, is reported here to be expressed in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. gas7 transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using in situ hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. Collectively, our results provide evidence for an association between expression of this gas gene and neuronal development.

Abstract

We identified a gene (dpiA, Destabilizer of Plasmid Inheritance) which, when overexpressed in Escherichia coli, destabilizes the inheritance of pSC101 and other iteron-containing plasmids as disparate as mini-F and RK6 but not the inheritance of P1, RSF1010 and ColD. These effects of DpiA, which functions like an effector protein for a previously undescribed two-component signal transduction system, were reduced by mutations known to promote pSC101 replication and partitioning. dpiB, a gene encoding the putative histidine kinase of this two-component system, is located immediately 5' to dpiA and adjacent to a DpiA-induced target promoter that transcribes genes having homology to citrate lyase operon genes, citC, citD and citE, of Klebsiella pneumoniae. Disruption of dpiB reversed or reduced the effect of DpiA overproduction on pSC101 inheritance. A second DpiA target, the promoter for a gene (appY) implicated in E. coli's response to anaerobiosis, is repressed by DpiA. A mutation in dpiA at a site commonly conserved and phosphorylated in two-component system effector proteins abolished the effects of DpiA overproduction on pSC101 inheritance and negative regulation of appY expression. Our findings suggest a possible mechanism by which environment and/or cellular stimuli may influence plasmid inheritance.

Abstract

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base -- providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.

Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiencyPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAXie, W. Q., Li, L. M., Cohen, S. N.1998; 95 (4): 1595-1600

Abstract

TSG101 is a recently discovered tumor susceptibility gene whose functional inactivation in mouse fibroblasts results in cell transformation and the ability to form metastatic tumors in nude mice. Although restoration of TSG101 activity reverses tumorigenesis, neoplasia is irreversible in some cells, suggesting that permanent genetic alteration can occur during TSG101 inactivation. Here we describe studies that support this notion. We find that localization of TSG101 is cell cycle-dependent, occurring in the nucleus and Golgi complex during interphase, and in mitotic spindles and centrosomes during mitosis; cells made neoplastic by a deficiency in TSG101 expression show a series of mitosis-related abnormalities, including multiple microtubule organizing centers, aberrant mitotic spindles, abnormal distribution of metaphase chromatin, aneuploidy, and nuclear anomalies. Our findings suggest that TSG101 deficiency may lead to genome instability in addition to previously reported reversible neoplastic transformation.

Abstract

Ribonuclease (RNase) E is an extensively studied enzyme from Escherichia coli whose site-specific endoribonuclease activity on single-stranded RNA has a central role in the processing of ribosomal RNA, the degradation of messenger RNA and the control of replication of ColE1-type plasmids. Here we report a previously undetected activity of RNase E: the ability to shorten 3' poly(A)- and poly(U)-homopolymer tails on RNA molecules. This activity, which leaves a 6-nucleotide adenylate or a 1-nucleotide uridylate remnant on primary transcripts, resides in the amino-terminal region of RNase E and does not require other protein cofactors. Addition of a 3'-terminal phosphate group prevents both removal of the poly(A) tail and endonucleolytic cleavage within primary transcripts, but has no effect on the cleavage of transcripts with tails that have already been truncated. The ability of RNase E to shorten poly(A) tails, together with the effect of tail length on endonucleolytic cleavage within primary transcripts, suggests a mechanism by which RNase E may exercise overall control over RNA decay.

Abstract

Intragenic deletions of TSG101, the human homolog of a mouse gene (tsg101) that acts to suppress malignant cell growth, were reported in human breast tumours. We screened TSG101 for somatic mutations in DNA and RNA samples isolated from a variety of common human malignancies, EBV-immortalised B-cells, and normal lung parenchyma. Intragenic TSG101 deletions in RNA transcripts were frequently found in all types of samples. Analysis of DNA failed to show genomic rearrangements corresponding to transcripts containing deletions in the same samples. The breakpoints of most transcript deletions coincide with genuine or cryptic splice site sequences, suggesting that they result from alternative or aberrant splicing. A similar spectrum of transcript deletions has previously been described in the putative tumour suppressor gene FHIT. We analysed FHIT in the same series of RNA samples and detected truncated FHIT transcripts frequently in both tumour and normal tissues. In addition, transcripts from TSG101, FHIT and seven other genes were analysed in RNA isolated from normal peripheral blood lymphocytes. Large TSG101 and FHIT intragenic transcript deletions were detected and these appeared to be the predominant transcript in 'aged' lymphocytes. Similar alterations were not detected in transcripts of the other genes which were analysed. Our findings demonstrate that truncated TSG101 and FHIT transcripts are commonly detected in both normal and malignant tissues and that a significant fraction of these are likely to be the result of aberrant splicing. While we cannot exclude that alterations in TSG101 and FHIT occur during cancer development, our data indicate that in this context the commonly observed transcript abnormalities are misleading.

Abstract

We report that the Streptomyces species S. lividans and S. coelicolor, morphologically complex gram-positive soil bacteria, contain a developmentally regulated endoribonuclease activity (here named RNase ES) that functionally and immunologically resembles Escherichia coli RNase E. In Streptomyces cells, RNA I - the antisense repressor of replication of ColE1-type plasmids - is cleaved at sites attacked by RNase E. A Mg2+-dependent endonuclease that produces RNase E-like cleavages in RNA I and 9S ribosomal RNA was identified in S. lividans cell extracts. A Streptomyces peptide migrating at 70kDa in SDS/polyacrylamide gels binds to RNase E substrates and reacts with three separate anti-RNase E monoclonal antibodies; the endonucleolytic cleavage activity co-purified with the immunoreactive 70 kDa peptide. We show that RNase ES activity is regulated during the Streptomyces life cycle: activity increased as cells progressed from exponential growth to stationary phase in liquid culture, or from mycelial growth to sporulation on solid media. While mutations that interfere with S. coelicolor development late in its life cycle did not prevent this developmentally associated increase in RNase ES activity, the increase was blocked by a mutation (bldA) that interferes early with both morphological and physiological differentiation.

Abstract

Ribonuclease E (RNase E), which is encoded by an essential Escherichia coli gene known variously as rne, ams, and hmp, was discovered initially as an rRNA-processing enzyme but it is now known to have a general role in RNA decay. Multiple functions, including the ability to cleave RNA endonucleolytically in AU-rich single-strand regions, RNA-binding capabilities, and the ability to interact with polynucleotide phosphorylase and other proteins implicated in the processing and degradation of RNA, are encoded by its 1,061 amino acid residues. The presence of homologues and functional analogues of the rne gene in a variety of prokaryotic and eukaryotic species suggests that its functions have been highly conserved during evolution. While much has been learned in recent years about the structure and functions of RNase E, there is continuing mystery about possible additional activities and molecular interactions of this enzyme.

Abstract

Recent work has identified a mouse gene (tsg101) whose inactivation in fibroblasts results in cellular transformation and the ability to produce metastatic tumors in nude mice. Here, we report that the human homolog, TSG101, which we isolated and mapped to chromosome 11, bands 15.1-15.2, a region proposed to contain tumor suppressor gene(s), is mutated at high frequency in human breast cancer. In 7 of 15 uncultured primary human breast carcinomas, intragenic deletions were shown in TSG101 genomic DNA and transcripts by gel and sequence analysis, and mutations affecting two TSG101 alleles were identified in four of these cancers. No TSG101 defects were found in matched normal breast tissue from the breast cancer patients. These findings strongly implicate TSG101 mutations in human breast cancer.

Abstract

Unlike previously studied linear replicons containing 5' DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA-binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single-stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.

The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) genePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALih, C. J., Cohen, S. N., Wang, C. Y., LINCHAO, S.1996; 93 (10): 4617-4622

Abstract

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

Abstract

We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.

Abstract

The rne gene of Escherichia coli encodes a 118 kDa protein that has ribonuclease E (RNase E) activity and binds RNA. A functional rne gene product is essential for cell viability and for the processing and/or decay of a variety of RNA species, including 9 S RNA, mRNA and RNAI, the antisense RNA regulator of ColE1-type plasmid replication. By testing the ability of different segments of the Rne protein to catalyze RNA cleavage and to bind RNA, we found that the N-terminal half (residues 1 to 498) of Rne contains a catalytic function sufficient for site-specific cleavage of oligoribonucleotides and complex RNAs. The C-terminal half of the protein, which contains both an arginine-rich region (residues 597 to 684) that we show binds RNA and a segment that is essential for cell viability (residues 844 to 1061), had no detectable endoribonucleolytic activity. Our results, which map the catalytic domain of RNase E, indicate the existence of discrete functional domains within the multifaceted Rne protein.

Abstract

Bovine papillomavirus (BPV) previously has been reported to exist in transformed rodent cell lines as both chromosomally integrated and extrachromosomal forms. In the BPV-transformed mouse cell line ID13, extrachromosomal BPV molecules replicate throughout S phase of the cell cycle in a random choice mode. We report here that these replication properties were altered for chromosomally integrated BPV DNA in five independent ID13 subclones. In all of the subclones, the integrated BPV sequences, which had no detectable deletions or mutations, existed in head-to-tail tandem arrays that replicated once per cell cycle, predominantly late in S phase. In contrast, extrachromosomal BPV molecules present in other subclones of the same cell line replicated in the random choice mode observed previously for non-integrated BPV. Our results indicate that the replication origin of integrated BPV either is inactivated as a consequence of chromosomal insertion, leading to the replication of BPV from origins in the flanking chromosomal DNA, or alternatively is reprogrammed to function in a once-per-cell cycle mode predominantly late in S phase.

Abstract

Rare cells that contain large amounts of bovine papilloma virus (BPV) DNA have been observed in populations of BPV-transformed mouse ID13 cells. The viral DNA molecules in these "jackpot cells" have been thought to have switched from the controlled replication typical of latent BPV infection to the uncontrolled "runaway" prelytic replication characteristic of terminal stage infection of bovine epidermal cells. By sequential subcloning of high-BPV derivatives of ID13, we isolated stable cell lines enriched more than 1000-fold for cells showing large amounts of BPV by fluorescence in situ hybridization analysis. High-BPV subclones contained a variant plasmid as well as wild-type BPV DNA; analysis of the BPV variants in two independently isolated subclones that showed the high-BPV phenotype in 50 to 80% of cells in the population indicated that both variants had undergone tandem duplication of the BPV long control region, which contains the viral origin of replication and transcription enhancer sequences. Transfer of the high-copy-number phenotype by transfection of DNA from high-BPV cells was accompanied by transfer of the variant plasmid. Density gradient analysis of BPV plasmid replication in high-BPV subclones showed the random-choice mode of replication observed in the parental ID13 population, rather than the rapid BPV replication found in epidermal cells destined for lysis and death. Our results indicate that high-BPV cells in actively dividing ID13 populations are not produced by uncontrolled replication of viral DNA and suggest that they may result instead from abnormal plasmid partitioning.

Abstract

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.

Abstract

The pSC101 RepA protein, which is required for plasmid DNA replication, but is inhibitory to replication at high concentration, has been found in both monomeric and dimeric forms. While RepA monomers bind to direct repeat iterons near the pSC101 replication origin, dimers bind to sequences that autoregulate RepA synthesis. We investigated the solution properties of purified RepA protein by analytical ultracentrifugation analysis, and found that RepA exists in Escherichia coli cells in a monomer-dimer equilibrium (Kd = 4 microM), and, moreover, that RepA is primarily in the monomeric form at the concentration (500 molecules per cell; 2 microM) we found by Western blot analysis to occur in cells carrying replicating wild-type pSC101 plasmids. However, at concentrations inhibitory to pSC101 DNA replication, the majority of RepA molecules exist as dimers. Our findings provide experimental support for the proposal that the equilibrium between monomer and dimer forms of RepA has a key role in determining its effect on the replication of pSC101.

Abstract

A 2.1-kb SacII DNA fragment of the high-copy-number Streptomyces lividans plasmid pIJ101 previously has been shown to include the functions required for maintenance of pIJ101 as an extrachromosomal replicon. This fragment contains the open translational reading frames rep and orf 56 plus an intervening segment believed to be noncoding. Using deletion mutations, we show that the pIJ101 replication origin and other cis-acting sites necessary and sufficient for replication map to a DNA segment that extends 515 bp 5' to the translational start of Rep and lacks orf56. Plasmids that include this segment are maintained in S. lividans as extrachromosomal replicons when complemented in trans by the rep gene product.

Abstract

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Abstract

Although polyadenylation has commonly been regarded as a special feature of eukaryotic messenger RNA, there are many reports of polyA tails on bacterial RNA (for example, refs 3-8). In Escherichia coli, adenylation mediated by the pcnB gene greatly accelerates decay of RNA I, an antisense repressor of replication of ColE1 type plasmids that resembles highly structured transfer RNA but shows the rapid turnover characteristic of mRNA. Here we report that both 3' adenylation and 5' phosphorylation affect the rate of digestion of RNA I by the 3' exonuclease, polynucleotide phosphorylase; conversely, mutation of the polynucleotide phosphorylase-encoding pnp gene affects ribonuclease acting at the 5' end. Together these findings indicate that enzymes attacking RNA I at its separate termini can interact functionally. Additionally, our discovery that adenylation-mediated degradation by polynucleotide phosphorylase imparts an mRNA-like half-like to RNA I suggests a possible mechanism to account for the rapid decay of mRNA in E. coli.

Abstract

Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells. We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication. Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA. A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity. Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons. Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function. Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA.

Abstract

Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene. While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability. Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding. Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions. They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.

Abstract

Earlier work has shown that the afsR genetic locus promotes formation of the pigmented antibiotics actinorhodin and undecylprodigiosin in Streptomyces lividans and its close relative, Streptomyces coelicolor. A protein designated as AfsR has been implicated in this activity. We report here the existence of a previously unknown gene, afsR2, which is separate from and adjacent to the AfsR-encoding sequence and which, when present at high copy number, (i) stimulates transcription of biosynthetic and regulatory genes in the actinorhodin gene cluster (act), and (ii) stimulates the synthesis of undecylprodigiosin. We show that the effects of afsR2 on actinorhodin synthesis are mediated through transcription of the actII-ORF4 locus, which encodes a transcriptional activator of other genes in the act cluster. Analysis of the cloned afsR2 gene indicates that its activity is the result of the 63-amino-acid protein it specifies.

ARD-1 - A HUMAN GENE THAT REVERSES THE EFFECTS OF TEMPERATURE-SENSITIVE AND DELETION MUTATIONS IN THE ESCHERICHIA-COLI RNE GENE AND ENCODES AN ACTIVITY PRODUCING RNASE E-LIKE CLEAVAGESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWang, M., Cohen, S. N.1994; 91 (22): 10591-10595

Abstract

The Escherichia coli rne gene affects a variety of bacterial functions, including the activity of RNase E. We report the existence of a human gene (ard-1, for activator of RNA decay) that complements temperature-sensitive and deletion mutations of rne in E. coli, allowing growth of rne-defective cells, correcting abnormal cell shape, activating chemical decay of bulk mRNA, and producing site-specific cleavages characteristic of RNase E in vivo and in vitro. ard-1 encodes a highly basic 13.3-kDa proline-rich peptide that has features in common with Rne and also with eukaryotic proteins implicated in RNA binding and macromolecular transport.

Abstract

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.

Abstract

RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end. The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain. Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E. coli and on cleavage of RNA I by RNase E in vitro. Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order. Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.

Abstract

Ribonuclease E has a central role in Escherichia coli mRNA decay and is dependent on a functional product of the rne (also called ams or hmp1) gene. We investigated the requirements for RNase E cleavage by introducing random mutations into the decanucleotide region at the 5' end of pACYC184 RNA I and studying the effects of these mutations on the position of rne-dependent cleavage in vivo and RNase E-mediated cutting in vitro. We find that the precise point of RNase E cleavage can be altered specifically and reproducibly by sequence changes in the region cleaved and, therefore, is not determined by a distance measured in nucleotides from any other sequence or region of secondary structure in RNA I. Although cleavage by RNase E occurs within sequences rich in A and/or U nucleotides and is affected by the extent of continuity of A and U nucleotides in the regions cleaved, there is no simple relationship between the order of nucleotides and the phosphodiester bond cleaved. Thus, our results are not consistent with either the notion that RNase E cleavages are determined by a simple consensus sequence or the contrary view that RNase E has few primary structural constraints other than a preference for cleaving 5' to an AU dinucleotide.

Abstract

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.

Abstract

Reviewed here are certain of the extraordinary contributions to molecular genetics that have resulted from the study of bacterial plasmids. Work with plasmids has led to both the 'operon' and 'replicon' concepts, and has provided seminal information about bacterial conjugation and fertility, recombination, transposable genetic elements, genome evolution and antisense RNA. Studies of plasmid functions have yielded important findings about the regulation of DNA replication, DNA topology and partitioning, gene control signals and restriction/modification enzymes. Plasmids have had a central role in the development of DNA cloning (recombinant DNA) methods; additionally, they have provided a paradigm for both the co-transformation of non-selected DNA into eukaryotic cells and the creation of the artificial chromosomes.

Abstract

RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division.

THE ACTIVE FORM OF THE KORB PROTEIN ENCODED BY THE STREPTOMYCES PLASMID PIJ101 IS A PROCESSED PRODUCT THAT BINDS DIFFERENTIALLY TO THE 2 PROMOTERS IT REGULATESJOURNAL OF BACTERIOLOGYTai, J. T., Cohen, S. N.1993; 175 (21): 6996-7005

Abstract

The korB gene of Streptomyces lividans plasmid pIJ101 is known to encode an autoregulated protein that also represses transcription of a gene, kilB, implicated in pIJ101 transfer and in spreading of the plasmid along mycelia of the recipient. Earlier work has indicated that the primary gene product of korB is a 10-kDa protein predicted from the gene sequence (D.S. Stein and S.N. Cohen, Mol. Gen. Genet. 222:337-344, 1990; S. Zamen H. Richards, and J. Ward, Nuleic Acids Res. 20:3693-3700, 1992). We report here that the 10-kDa KorB protein product is processed in vivo into a 6-kDa peptide that has a 20-fold-greater binding affinity for its operator-promoter target; in addition, the 6-kDa peptide binds differentially to the regulatory regions of the two genes it controls, showing 50-fold-greater affinity for the kilB sequence. While both the processed and unprocessed forms of KorB were observed in Escherichia coli following korB gene expression under control of the bacteriophage T7 promoter, only the 6-kDa peptide was found in S. lividans containing pIJ101, implying that this peptide is normally the biologically active form of KorB. The footprint resulting from KorB binding to the korB operator sequence overlaps the sti locus, which affects pIJ101 copy number and incompatibility as well as the size of zones of inhibited recipient cell growth ("pocks") that form around donor cells during mating. The observed ability of the korB gene product to interact with both sti sequences and the kilB promoter region suggests that it may have a role in coordinating the replication and intramycelial spread of plasmids during and/or following bacterial mating.

Abstract

The Streptomyces coelicolor genetic element SLP1 can exist either integrated into the host chromosome or as an autonomously replicating plasmid. The integrated form of SLP1 includes a locus (imp, for inhibition of plasmid maintenance) that can act both in cis and in trans to prevent propagation of SLP1 as an extrachromosomal replicon (S. R. Grant, S. C. Lee, K. Kendall, and S. N. Cohen, Mol. Gen. Genet. 217:324-331, 1989). We report here that a 1.8-kb Eco47III DNA fragment previously shown to encode the Imp+ phenotype contains two genes (impA and impC) that must be expressed in cis to each other and whose products interact functionally and probably physically to interfere with SLP1 plasmid maintenance and repress expression of the imp operon. Partial repression of the imp promoter (P(imp)), which is located immediately 5' of impA, by the 29.7-kDa ImpA protein is enhanced by the impC gene product. Gel shift analysis indicates that ImpA binds to a 16-bp sequence located within the DNA segment containing P(imp) and that ImpC interferes with this binding. Our data suggest that binding of ImpA to the P(imp) region mediates DNA looping in this region.

Abstract

We report here direct evidence that mutations in the par locus affect protein-DNA interactions in vivo at the replication origin of plasmid pSC101. Concomitant with par-mediated plasmid stabilization, two sites in the origin region show an altered methylation pattern as detected by in vivo footprinting with dimethyl sulfate. One site is located near an integration host factor-binding sequence adjacent to the first of three direct repeats known to be involved in the initiation of pSC101 replication; the second site is within the third direct repeat.

Abstract

The incompatibility that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superhelicity, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid. We propose a model by which the par locus can enhance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.

THE ESCHERICHIA-COLI PCNB GENE PROMOTES ADENYLYLATION OF ANTISENSE RNAI OF COLE1-TYPE PLASMIDS IN-VIVO AND DEGRADATION OF RNAI DECAY INTERMEDIATESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAXu, F. F., LINCHAO, S., Cohen, S. N.1993; 90 (14): 6756-6760

Abstract

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.

THE AMS-1 AND RNE-3071 TEMPERATURE-SENSITIVE MUTATIONS IN THE AMS GENE ARE IN CLOSE PROXIMITY TO EACH OTHER AND CAUSE SUBSTITUTIONS WITHIN A DOMAIN THAT RESEMBLES A PRODUCT OF THE ESCHERICHIA-COLI MRE LOCUSJOURNAL OF BACTERIOLOGYMcDowall, K. J., Hernandez, R. G., Chao, S. L., Cohen, S. N.1993; 175 (13): 4245-4249

Abstract

Two temperature-sensitive mutations, ams-1 and rne-3071, in the ams (altered mRNA stability) gene have been used extensively to investigate the processing and decay of RNA in Escherichia coli. We have sequenced these temperature-sensitive alleles and found that the mutations are separated by only 6 nucleotides and cause conservative amino acid substitutions next to a possible nucleotide-binding site within the N-terminal domain of the Ams protein. Computer analysis revealed that the region altered by the mutations has extensive sequence similarity to a predicted gene product from the mre (murein pathway cluster e) locus of E. coli, which has been implicated previously in determining bacterial cell shape.

Abstract

The beta satellite sequences of the human genome are a family of genetic elements consisting of 68-69 bp monomeric units repeated contiguously in long arrays up to 1 Mb in length. We have determined the timing of replication of beta satellite subgroups located in the heterochromatic portion of chromosome 9 and on the acrocentric chromosomes in regions both distal and proximal to the rDNA genes. We report that these dispersed subgroups of beta satellite sequences all replicate late during S phase of the cell cycle.

Abstract

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.

EXCISIVE RECOMBINATION OF THE SLP1 ELEMENT IN STREPTOMYCES-LIVIDANS IS MEDIATED BY INT AND ENHANCED BY XISJOURNAL OF BACTERIOLOGYBrasch, M. A., Cohen, S. N.1993; 175 (10): 3075-3082

Abstract

The functions mediating site-specific recombination of the SLP1 element have been mapped to a 2.2-kb region that includes the site of integration (attP), a gene (int) that specifies a function both necessary and sufficient for integration of SLP1, and an open reading frame, orf61, suspected of encoding a protein, Xis, that shows limited similarity to the excisionases of other site-specific recombination systems. Here we describe experiments that investigate the respective roles of orf61 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pIJ101 that express orf61, int, or both orf61 and int from transcriptional fusions to the Tn5 aph gene and tested the ability of these constructs to promote excision of an adventitious attP-containing plasmid that had been integrated site-specifically into the attB site of the Streptomyces lividans chromosome. Expression of the int gene product alone from an exogenous promoter was sufficient for excision of the integrated plasmid. This result indicates that the SLP1 int-encoded protein can carry out excisive, as well as integrative, recombination. The orf61 gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal attB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the orf61-encoded protein. On the basis of these findings, we here designate the orf61 gene as xis.

Abstract

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.

Abstract

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.

RECONSTRUCTION OF A STREPTOMYCES LINEAR REPLICON FROM SEPARATELY CLONED DNA FRAGMENTS - EXISTENCE OF A CRYPTIC ORIGIN OF CIRCULAR REPLICATION WITHIN THE LINEAR PLASMIDPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAShiffman, D., Cohen, S. N.1992; 89 (13): 6129-6133

Abstract

We report here the reconstruction of a functional linear replicon, the 12-kilobase Streptomyces clavuligerus plasmid pSCL, from separate DNA fragments cloned in Escherichia coli on the pUC19 plasmid. Protein-free DNA molecules containing the full-length pSCL sequence, an internally inserted thiostrepton-resistance gene, and adventitious nucleotides external to the pSCL termini were introduced into Streptomyces lividans, where the synthesis and functional attachment of replication proteins occurred and pSCL was established as an extrachromosomal linear replicon. Transformation of S. lividans with uncut supercoilded pUC19/pSCL DNA from E. coli or with a circularized 8-kilobase internal fragment of pSCL yielded circular replicons, indicating the existence of a cryptic origin of circular replication within the linear plasmid. Insertion mutations at sites that prevented the replication of pSCL linear plasmids also interfered with its replication in the circular mode.

Abstract

Integration host factor (IHF), a multifunctional protein of E. coli, normally is required for the replication of plasmid pSC101. T. T. Stenzel, P. Patel, and D. Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate. We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter. With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself. These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex. A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex.

Abstract

Segments of the murine genome that hybridize to the inverted repeat regions of the transposable TU elements of sea urchins include tandem repeats of a sequence (CTCC) that encodes the recognition site for the restriction enzyme Mnl1, as do the analogous polypurine/polypyrimidine (pPu/pPy) stretches in humans. The Mnl1-sensitive repeats, which exist as a microsatellite sequence 200-300 bp in length, lack the terminal dyad symmetry characteristic of the TU elements and are structurally and functionally distinct from these elements. DNA fragments containing these repeat units that are isolated from different generations of isogenic (or congenic) mice or from different tissues of genetically identical individuals are indistinguishable by RFLP analysis; however, they show restriction fragment length polymorphism in different strains. This polymorphism appears to reflect DNA sequence changes occurring at sites flanking the repeats rather than variability in the number of repeats. Their genetic stability and occurrence in a wide variety of animal species make the Mnl1 repeats useful in studying genetic variation that has occurred over an evolutionary time scale of greater duration than can be examined conveniently by VNTR analysis.

Abstract

The pSC101 plasmid encodes a cis-acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells. In the absence of selection, par-defective plasmids are lost rapidly from the bacterial population. We report here that the stability of par-deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid. Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity. While a promotor-associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling. Our demonstration that promoter-induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity-generating effects of par are intrinsic to its function.

Abstract

We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number.

Abstract

Previous work has shown that expression of genes within the polycistronic puf operon of Rhodobacter capsulatus is regulated in part by differential degradation of segments of puf transcripts. To understand the role of ribosome coverage in the differential stability of puf mRNA segments, we have studied the effects of mutations that alter translation of specific puf transcript segments on puf mRNA decay. Our results show that stopping translation either within the light-harvesting I (LHI) genes or near the 5' end of the reaction center (RC)-coding region decreased the stability of puf transcript segments downstream from a hairpin loop structure located between the LHI and RC genes but failed to affect the upstream sequences so long as the loop was present. Mutations that allowed translation to proceed through the hairpin structure reduced its ability to protect upstream sequences from accelerated decay. Introduction of translation stops more than 107 bp into the RC-coding region, but still 5' to an mRNA segment containing decay-promoting endonuclease cleavage sites, had no effect on puf mRNA stability. The divergent and location-dependent consequences of translation stops imply that different mechanisms are responsible for the degradation of different puf mRNA segments and indicate that coverage of puf mRNA sequences by ribosomes is insufficient and may in some cases be unnecessary to protect these sequences from degradation.

Abstract

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.

Abstract

We describe a method for the rapid two-stage amplification and detection by ethidium bromide staining of chromosomal nucleotide (nt) sequences in lysates made directly from anchorage-dependent cells attached to microcarrier beads. The procedure circumvents the need for cell detachment steps prior to analysis, facilitates the collection, transfer, and manipulation of the cells being studied, and makes unnecessary the use of Southern-blot hybridization for identification of specific nt sequences present in a small fraction of cells within a heterogeneous population.

Abstract

Differential expression of the genes within the puf operon of Rhodobacter capsulatus is accomplished in part by differences in the rate of degradation of different segments of the puf transcript. We report here that decay of puf mRNA sequences specifying the light-harvesting I (LHI) and reaction center (RC) photosynthetic membrane peptides is initiated endoribonucleolytically within a discrete 1.4-kilobase segment of the RC-coding region. Deletion of this segment increased the half-life of the RC-coding region from 8 to 20 min while not affecting decay of LHI-coding sequences upstream from an intercistronic hairpin loop structure shown previously to impede 3'-to-5' degradation. Prolongation of RC segment half-life was dependent on the presence of other hairpin structures 3' to the RC region. Inserting the endonuclease-sensitive sites into the LHI-coding segment markedly accelerated its degradation. Our results suggest that differential degradation of the RC- and LHI-coding segments of puf mRNA is accomplished at least in part by the combined actions of RC region-specific endonuclease(s), one or more exonucleases, and several strategically located exonuclease-impeding hairpins.

Abstract

The N-terminal domains of the alpha and beta polypeptides of the B870 antenna complex of Rhodobacter capsulatus are oppositely charged. In both polypeptides two charged amino acids are located close to the N-terminus, and two of them are close to the hydrophobic central domain. To test the hypothesis that charged amino acids in the N-terminus have a function for insertion and assembly of pigment-binding polypeptides, charged amino acids were replaced by amino acids of opposite charge. The results show that an exchange of amino acid positions 3 and 6 in alpha (Lys----Glu) or 2 and 5 in beta (Asp----Lys, Arg) has little effect under semiaerobic conditions on the formation of B870 but the additional exchange of positions 14 and 15 in alpha (Arg----Glu, Asp) and/or 13 and 14 in beta (Asp, Glu----Arg) inhibits strongly under semiaerobic dark and anaerobic light conditions the stable incorporation of the polypeptides into the membrane and the formation of the B870 complex. The mutant U43(pTXAB5) is able to grow without any antenna.

Abstract

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.

Abstract

cis-acting mutations that affect regulation of the Rhodobacter capsulatus puf operon by oxygen were isolated by placing the mutagenized puf regulatory region 5' to a promoterless Tn5 neo gene, which encodes resistance to kanamycin (Kmr). R. capsulatus mutants that failed to show wild-type repression of KMr by oxygen were selected and analyzed. Four independent clones contained point mutations, three of which were identical, in a region of dyad symmetry located between puf operon nucleotide positions 177 and 207, approximately 45 base pairs 5' to the site of initiation of puf transcripts. The phenotypic effects of the aerobically selected mutations were duplicated by single and double point mutations introduced site specifically into the region of dyad symmetry by oligonucleotide-directed mutagenesis. Determinations of the bacterial 50% lethal dose of kanamycin, of aminoglycoside phosphotransferase activity in cell sonicates, and of neo-specific mRNA confirmed the diminished responsiveness of the mutants to oxygen and consequently implicated the mutated region in O2-mediated transcriptional regulation.

Abstract

Previous work has shown that a cis-acting locus (termed par for partitioning) on the pSC101 plasmid accomplishes its stable inheritance in dividing cell populations. We report here that the DNA of pSC101 derivatives lacking the par region shows a decrease in overall superhelical density as compared with DNA of wild-type pSC101. Chemicals and bacterial mutations that reduce negative DNA supercoiling increase the rate of loss of par plasmids and convert normally stable plasmids that have minimal par region deletions into unstable replicons. topA gene mutations, which increase negative DNA supercoiling, reverse the instability of partition-defective plasmids that utilize the pSC101, p15A, F, or oriC replication systems. Our observations show that the extent of negative supercoiling of plasmid DNA has major effects on the plasmid's inheritance and suggest a mechanism by which the pSC101 par region may exert its stabilizing effects.

Abstract

The korA and korB loci of Streptomyces lividans plasmid pIJ101 have previously been shown to control expression of the pIJ101 tra (formerly kilA) and kilB genes at the transcriptional level. We show here that mutations in translational open reading frames (ORFs) that map within the kor loci abolish repression of the S. lividans lac gene directed by the tra and kilB promoters. Introduction of the korA and korB ORFs into Escherichia coli maxicells under control of an E. coli promoter gave rise to 31 kDa and 10 kDa proteins that correspond in size to the products expected from the sequences of the respective ORFs; these proteins controlled transcription from the pIJ101 tra and kilB promoters in the E. coli host. Mutations that affected the KorA or KorB phenotype altered the structure of, or eliminated, the protein products of the korA and korB ORFs, further demonstrating that these ORFs encode the functional repressors of the pIJ101 kil/kor gene system.

Abstract

We describe the characteristics of pro-opiomelanocortin (POMC) mRNA synthesized by a human small cell lung cancer (SCLC) cell line that secretes a peptide immunoreactive with antibodies to the POMC-derived component, adrenocorticotropin. While no alteration in restriction endonuclease pattern or structure was found for the SCLC-derived pomc gene vs. the previously described human pomc gene cloned from a fetal liver library, Northern-blot analysis of SCLC RNA using pomc-derived probes showed a hybridizing transcript more than 300 nucleotides longer than POMC mRNA isolated from human pituitaries, as well as a pomc-gene-hybridizing mRNA the same length as pituitary-derived transcripts. 5' end mapping and primer extension analyses showed that the novel mRNA species is initiated at a site 371 bp upstream from the 5' end identified for pituitary-derived POMC mRNA. We conclude that synthesis of POMC transcripts occurs from an ordinarily quiescent promoter in the SCLC cell line we have studied, as well as from the pomc promoter normally used in pituitary cells.

Abstract

Four regulated promoters that direct the transcription of genes (i.e., korA, tra, kilB, and korB) involved in the transfer of the Streptomyces plasmid pIJ101 were isolated following the in vitro fusion of plasmid DNA fragments to a promoterless gene encoding the S. lividans extracellular enzyme beta-galactosidase. Introduction of pIJ101 into cells carrying each of these promoter-lac fusions resulted in decreased lac expression. The sites of initiation of transcription by the promoter sequences were identified by primer extension experiments, and the DNA sequences specifically required for promoter activity and regulation by pIJ101-encoded functions were determined by deletion analysis. The data obtained indicate that the korB locus encodes a repressor that regulates its own transcription, as well as transcription of the kilB promoter; korA and tra are transcribed from overlapping divergent promoters that are coregulated by the korA gene product. Common DNA sequence domains within coregulated promoters allowed the identification of putative binding sites for each of the kor gene products.

ANALYSIS OF MAMMALIAN-CELL GENETIC-REGULATION INSITU BY USING RETROVIRUS-DERIVED PORTABLE EXONS CARRYING THE ESCHERICHIA-COLI LACZ GENEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABrenner, D. G., Chao, S. L., Cohen, S. N.1989; 86 (14): 5517-5521

Abstract

Self-inactivating derivatives of Moloney murine leukemia retrovirus containing the Escherichia coli lacZ gene were used to detect and study the regulation of transcription initiated at chromosomally located promoters in mouse fibroblasts. The introduction of splice acceptor sites in all three translational reading frames relative to lacZ and the inclusion of an in-frame ATG translation start codon in one construct allowed synthesis of beta-galactosidase fusion proteins upon insertion of retrovirus vectors containing lacZ into introns 3' to either protein-coding or noncoding exons. Selection of lacZ-expressing cells by fluorescence-activated cell sorting and the analysis of beta-galactosidase production after serum deprivation has yielded lines in which lacZ was fused to genes induced by growth arrest in the G0 state.

Abstract

We report here the existence and initial characterization of a genetic locus (imp) that inhibits maintenance of SLP1-derived plasmids as extrachromosomal replicons in a manner distinct from normal incompatibility between autonomous SLP1 replicons. The trans-acting imp function has been localized to a 1.8 kb Eco47III restriction fragment present on integrated SLP1 elements. At least part of this DNA segment is absent from SLP1-derived plasmids. DNA sequence analysis of the imp region indicates that it contains three overlapping open reading frames (ORFs) that may constitute a polycistronic operon. The effects of insertions within the imp region indicate that uninterrupted transcription through all three ORFs is necessary for imp activity.

Abstract

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.

Abstract

We report here the cloning of a Streptomyces lividans gene that when introduced on a multicopy plasmid vector reversed the pigment deficiency phenotype of several distinct mutants blocked in development, pigment production, or both. Although this gene was shown by restriction enzyme analysis to be similar to a previously cloned afsB-complementing gene of Streptomyces coelicolor, we show that it does not correspond to the S. coelicolor chromosomal locus designated afsB. Thus, the cloned locus, which we propose to rename afsR, appears to complement the AfsB- phenotype by pleiotropic regulatory effects.

Abstract

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.

Abstract

We report data indicating that the Rhodobacter capsulatus puf operon promoter and the site for its oxygen regulation are located more than 700 base pairs upstream from the previously identified puf genes and have identified the nucleotide sequences that constitute these control signals. A model is proposed in which a polycistronic transcript at least 3.4 kilobases in length is initiated near the O2-regulated promoter and is processed posttranscriptionally by endonucleolytic cleavage at multiple sites, yielding discrete mRNA segments that are degraded at different rates. A newly identified gene (pufQ), which includes a hydrophobic domain having some similarity to domains of the products of the pufL and pufM genes, begins 313 nucleotides into the puf transcript and is located entirely within the most rapidly degraded segment of the transcript. A previously identified puf transcript segment encoding structural proteins for photosynthetic membrane complexes persists after degradation of the most 5' region of the transcript and is itself subject to segmentally specific degradation. Our results suggest a model in which differential expression of the multiple genes encoded by the puf operon is at least in part attributable to major differences in the rates of decay of the various segments of puf mRNA.

Abstract

SLP1int is a conjugative Streptomyces coelicolor genetic element that can transfer to Streptomyces lividans and integrate site specifically into the genome of the new bacterial host. Recombination of SLP1 previously has been shown to occur within nearly identical 112-base-pair att sequences on the plasmid and host chromosome. We report here that both integrative recombination and intermolecular transfer of SLP1int require no more than a 48-base-pair segment of the att sequence and that SLP1 transfer occurs by a conservative rather than a replicative mechanism. The functions responsible for the excision of the element as a discrete DNA segment are induced during the conjugal transfer of SLP1.

Abstract

The polycistronic puf operon of Rhodobacter capsulatus encodes protein components for the photosynthetic reaction center and one of the two antenna complexes involved in the capture of light energy. We report here that deletions within specific puf genes alter the synthesis and/or assembly in the photosynthetic membranes of pigment-protein complexes not affected genetically by the deletion. The pufX gene is required for normal ratios of antenna complexes, and its deletion results in an increase of membrane-bound light-harvesting I (LHI) complex-specific proteins. Expression of pufQ in strains deleted for the genes encoding the LHI and the photosynthetic reaction center (RC) yields a novel A868 peak that has not been associated with any of the pigment-protein complexes described previously. While deletions in the RC-coding region resulted in decreased LHI absorbance, no quantitative alteration in membrane-bound LHI protein was observed, suggesting that an intact RC complex is required for correct assembly of LHI in the membrane.

Abstract

The complete nucleotide sequence of the multicopy Streptomyces plasmid pIJ101 has been determined and correlated with previously published genetic data. The circular DNA molecule is 8,830 nucleotides in length and has a G+C composition of 72.98%. The use of a computer program, FRAME, enabled identification in the sequence of seven open reading frames, four of which, tra (621 amino acids [aa]), spdA (146 aa), spdB (274 aa), and kilB (177 aa), appear to be genes involved in plasmid transfer. At least two of the above genes are predicted to be transcribed by known promoters that are regulated in trans by the products of the korA (241 aa) and korB (80 aa) loci on the plasmid. The segment of the plasmid capable of autonomous replication contains one large open reading frame (rep; 450 aa) and a noncoding region presumed to be the origin of replication. Four other small (less than 90 aa) open reading frames are also present on the plasmid, although no function can be attributed to them. The sequence of the pIJ101 replication segment present in several widely used cloning vectors (e.g., pIJ350 and pIJ702) has also been determined, so that the complete nucleotide sequences of these vectors are now known.

Abstract

We report that transformation of Streptomyces lividans with cloned DNA of the SLP1 genetic element results in integration of the element at the same chromosomal locus (attB) normally occupied by SLP1 in its original host, Streptomyces coelicolor, and in S. lividans that has received SLP1 by mating. We constructed SLP1 derivatives that can integrate foreign DNA at the attB site and used these to introduce adventitious DNA sequences into the S. lividans chromosome. We also identified three regions of SLP1 essential for its integration and demonstrated that integration of the SLP1 element does not require expression of functions necessary for stable maintenance or transfer of extrachromosomal forms of SLP1.

Abstract

Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capsulatus. The comparatively stable 5' segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5' puf mRNA segment but that its insertion at the 3' end of the puf operon transcripts fails to stabilize the labile 3' puf mRNA segment. Evidence is presented that decay of the 3' segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3' exoribonucleases.

Abstract

It has been proposed that intercistronic stem and loop structures located in the puf operon of the photosynthetic bacterium Rhodobacter capsulatus account for segmental differences in transcript stability and consequently, the differential expression of the B870 and reaction center (RC) proteins encoded by puf. We report here that deletion of these structures leads to a failure to detect as discrete fragments the B870-encoding 0.49 kb and 0.50 kb mRNA segments located upstream from the site of the hairpins. The absence of these stable transcript fragments is associated with altered stoichiometry of the B870 and RC pigment-protein complexes in the bacterial intracytoplasmic membrane and a decreased rate of cell growth under photosynthetic conditions. These results support the view that the hairpin loop structures of the puf intercistronic region function in vivo to impede exoribonucleolytic degradation of upstream mRNA and establish that segmental variations in mRNA stability have a biologically important role in regulating the expression of puf operon genes.

Abstract

The 8.9-kilobase Streptomyces plasmid pIJ101 is self-transmissible at high frequency into recipient strains. By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal pIJ101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB. KilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever. Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively. These results suggest that kilA, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes.

Abstract

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.

EFFECT OF PREMATURE TERMINATION OF TRANSLATION ON MESSENGER-RNA STABILITY DEPENDS ON THE SITE OF RIBOSOME RELEASEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANilsson, G., Belasco, J. G., Cohen, S. N., VONGABAIN, A.1987; 84 (14): 4890-4894

Abstract

Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed. Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments. Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA. These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation. However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript.

Abstract

We previously have shown that homologs of the outer domain segment of the inverted repeat termini (IVR-OD) of the sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. We report here that two cloned human DNA IVR-OD homologs, Hut2 and Hut17, consist of a series of tandem repeats of the trimer AGG/TCC, forming segments (313 and 221 base pairs in length, respectively) of polypurine/polypyrimidine (pPu/pPy or "Puppy") asymmetry in the two DNA strands; these are punctuated at certain sites with variant trimers, which are different for the two clones. Sequences homologous to the Hut2 pPu/pPy tract exist at multiple sites in the DNA of a wide variety of eucaryotes. Hybridization of human DNA with a Hut2 probe or with a previously described chicken DNA pPu/pPy sequence indicates that pPu/pPy sequences can be grouped into families distinguishable by the extent of their homology with each probe at different hybridization stringencies. Moreover, particular pPu/pPy tracts show species-specific differences in their distribution. Both the Hut2 and Hut17 pPu/pPy tracts are cleaved by S1 nuclease when tested on supercoiled plasmids. Most if not all of the 313-base-pair Hut2 pPu/pPy tract is also sensitive to S1 in its native location in HeLa cell chromatin, indicating that the sequence contains conformational information that can be expressed in vivo. This view is supported by evidence that exogenously derived Hut2 pPu/pPy tracts introduced into mouse L cells and integrated in chromatin can assume an S1-sensitive conformation.

Abstract

We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme.

Abstract

To map the structural features responsible for the 5-fold difference in stability of the E. coli ompA and bla gene transcripts, we have constructed gene fusions that encode chimeric ompA/bla transcripts and a deletion that eliminates a large internal segment of bla mRNA. Shortening of bla transcripts by internal deletion or replacement of the 3' end with the corresponding segment of the ompA transcript had little effect on bla mRNA stability. However, fusion of a 5'-terminal 147 nucleotide segment of the ompA message 5' to full-length or truncated bla transcripts increased the half-life of the bla segments 3- to 5-fold. These and other findings indicate that E. coli transcripts contain discrete structural determinants of stability and instability that can influence the decay rate of linked mRNA segments derived from other genes.

Abstract

SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes.

Abstract

Messenger RNA was extracted from porcine pituitary pars intermedia and cloned as cDNA by standard methods. The nucleotide sequence encoding porcine pro-opiomelanocortin was established from analysis of two separate cDNA segments having an overlap of 420 bases. The amino acid sequence for the porcine pro-opiomelanocortin protein, which was inferred from the cDNA sequence, was found to correspond exactly to the sequence determined by direct amino acid analysis of the component peptides of pro-opiomelanocortin; namely, the porcine hormones ACTH, beta-lipotropin, gamma-MSH and the connecting peptide. We thus find no evidence for the existence of two porcine pro-opiomelanocortin genes that differ in their coding sequences, as was suggested by the in vitro protein synthesis results of others using mRNA obtained from porcine pituitaries.

Abstract

Sequences homologous to various structural domains of the Strongylocentrotus purpuratus TU family of transposons are present in sea urchin species closely related to S. purpuratus and were found in close proximity to each other in linkage patterns that differed for different species. Sequence homologs of the inverted repeat outer domain (IVR-OD) segment were, in addition, present in a sea urchin related only distantly to S. purpuratus and in all other eucaryotic organisms surveyed. In humans, a polymorphic hybridization pattern was seen for genomic DNA obtained from different individuals. Sequence comparisons revealed that repeated sequence motifs similar to those making up the 15-base-pair direct repeat unit of the IVR-OD domain of the TU elements exist in the IVRs of transposons identified in Drosophila melanogaster and maize and in the transcription control regions of certain eucaryotic viral and cellular genes. The remarkable evolutionary conservation of IVR-OD homologs may reflect a biological role for these sequences in DNA transposition, the regulation of gene expression, or both.

Abstract

Promoter-probe plasmid vectors were used to isolate putative promoter-containing DNA fragments of three Streptomyces antibiotic resistance genes, the rRNA methylase (tsr) gene of S. azureus, the aminoglycoside phosphotransferase (aph) gene of S. fradiae, and the viomycin phosphotransferase (vph) gene of S. vinaceus. DNA sequence analysis was carried out for all three of the fragments and for the protein-coding regions of the tsr and vph genes. No sequences resembling typical E. coli promoters or Bacillus vegetatively-expressed promoters were identified. Furthermore, none of the three DNA fragments found to be transcriptionally active in Streptomyces could initiate transcription when introduced into E. coli. An extremely biased codon usage pattern that reflects the high G + C composition of Streptomyces DNA was observed for the protein-coding regions of the tsr and vph genes, and of the previously sequenced aph gene. This pattern enabled delineation of the protein-coding region and identification of the coding strand of the genes.

Abstract

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.

Abstract

We describe here a family of foldback transposons found in the genome of the higher eucaryote, the sea urchin Strongylocentrotus purpuratus. Two major classes of TU elements have been identified by analysis of genomic DNA and TU element clones. One class consists of largely similar elements with long terminal inverted repeats (IVRs) containing outer and inner domains and sharing a common middle segment that can undergo deletions. Some of these elements contain insertions. The second class is highly heterogeneous, with many different middle segments nonhomologous to those of the first-class and variable-sized inverted repeats that contain only an outer domain. The middle and insertion segments of both classes carry sequences that also are found unassociated from the inverted repeats at many other genomic locations. We conclude that the TU elements are modular structures composed of inverted repeats plus other sequence domains that are themselves members of different families of dispersed repetitive sequences. Such modular elements may have a role in the dispersion and rearrangement of genomic DNA segments.

Abstract

We describe here the isolation and characterization of a class of A + T-rich transcriptionally active sequences in the filamentous antibiotic-producing Gram-positive bacterial genus Streptomyces. These regions, which digress dramatically in base composition from the 73% G + C composition characteristic of the Streptomyces genome, promote gene expression in both Escherichia coli and Streptomyces lividans and contain the major elements that determine promoter strength in E. coli. The Streptomyces-E. coli-type promoters (SEP) also show novel structural features that include multiple direct repeats within the promoter region as well as a specific hexameric sequence in the vicinity of the mRNA start point.

Abstract

We report that the light-harvesting and reaction center genes in the rxcA locus of R. capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript. The 3' portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides. The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins. The unstable 3' portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3' exoribonucleases and thereby protect the upstream RNA segment. When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E. coli, the long precursor transcript was processed to two short messages of greater stability, as in R. capsulata.

Abstract

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-to-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.

Abstract

We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) originate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragments of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.

Abstract

The rate of production of bacterial gene products is known to vary with the rate of cell growth, the concentrations of many cellular proteins are altered during times of decreased growth rate. In addition, proteins whose in vivo levels show no significant alterations with changes in cell doubling time must be synthesized at rates that vary in direct proportion to the growth rate of the cell. In certain instances, growth-rate dependent gene regulation has been shown to occur at the transcriptional or translational level. Another potentially important element in the regulation of gene expression is the stability of messenger RNA. We report here the effect of bacterial growth rate on the half lives of four different monocistronic Escherichia coli mRNA species. The stabilities of two of these species, the transcripts of the ompA and cat genes, exhibited a marked dependence on cell growth rate, whereas the half lives of the transcripts of the lpp and bla genes are constant over a broad range of cell doubling times. Our results indicate that E. coli can alter the rate of synthesis of certain proteins by modulating mRNA stability in response to changes in the rate of cell growth.

Abstract

We report here the use of a novel genetic approach for the study of transcriptional control in Streptomyces lividans. Using up-promoter mutants of the ampC beta-lactamase gene of Escherichia coli, we have shown that mutations in each of the regions that define the major determinants of promoter strength in E. coli-i.e., the -35 region, the -10 region, and the intervening "spacer" region-lead to increased synthesis of ampC mRNA in S. lividans, just as they do in their host of origin. Results are also presented showing that the ampC transcriptional terminator is functional in S. lividans. Taken together, these findings indicate that Streptomyces have an RNA polymerase that recognizes and uses the various components of E. coli transcriptional signals, and imply that, notwithstanding the high GC content (i.e., 73%) of the Streptomyces genome, these organisms have indigenous promoters similar to those found in E. coli.

Abstract

Three distinct segments (the partition-related, or PR segments) within the 370 bp par region of pSC101 have been shown by deletion analysis to be involved in partitioning of the plasmid to daughter cells. The two lateral segments are direct repeats, each of which potentially can pair with an inverted repeat located between them to form a hairpin-loop structure. Deletion of either lateral segment, together with the middle segment, results in plasmid instability (the Par- phenotype). Deletion of one PR segment yields a stable plasmid that nevertheless shows reduced ability to compete with a coexisting wild-type derivative of the same replicon (the Cmp- phenotype). Deletion of all three segments results in a rate of plasmid loss far in excess of that predicted from the observed copy number of the plasmid. Analysis of the segregation properties of these mutants and of temperature-sensitive and high copy number derivatives of the pSC101 replicon suggests a model in which the par function allows the nonreplicating plasmids of the intracellular pool to be counted as individual molecules, and to be distributed evenly to daughter cells. In the absence of par, the multicopy pool of plasmids behaves as a single segregation unit.

Abstract

Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoid volume forming a tissue-like mass. Production rates of beta-lactamase, an enzyme used as an indicator of the culture's biosynthetic potential, remained at high and relatively stable levels for more than three weeks of continuous operation, and effluent supernatant enzyme activities attained 25% of the accumulated level measured in a 24-h shaker-flask culture. Based on the accessible void volume within the fiber wall, the beta-lactamase productivity was independent of the specific asymmetric membrane used. On a per cell basis, however, cells cultured using hollow-fiber membranes were only 10% as productive as those in the shaker-flask culture, possibly due to the high packing density or culture aging. By contrast, the hollow-fiber bioreactor was 100 times more productive than the shaker-flask culture on a reactor-volume basis, primarily as a consequence of the high cell densities. Reactor productivity was dependent on the number of cells in the reactor, suggesting that reactor performance was kinetically controlled and not mass transport limited.

Abstract

We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.

Abstract

Cellular DNAs from a panel of 20 unrelated individuals were screened for restriction fragment length polymorphisms (RFLP) with a DNA probe containing the first exon of the proopiomelanocortin gene (POMC), which has been assigned to chromosome 2p23-25. Digestion with the restriction endonuclease Sst 1 revealed a high frequency RFLP. The two alleles that were found are fragments of 10- and 15-kilobase (kb) length and are in Hardy-Weinberg equilibrium with frequencies of 72.6% and 27.4%, respectively. Informative families were tested for linkage between POMC/Sst 1 RFLP and other polymorphic markers of chromosome 2. Linkage was excluded to AcP-1 (2p23-25) at 15% recombination, which is still consistent with the chromosomal assignments for these genes. The close physical linkage (10 kb) of the polymorphic locus to the POMC gene makes this RFLP a suitable marker for future linkage studies involving the POMC gene.

Abstract

The restriction map of a 46-kilobase fragment of the Rhodopseudomonas capsulata chromosome was aligned with the genetic map of the photosynthesis region of that chromosome by a marker rescue technique. Marker rescue was effected by mobilization of vectors bearing fragments of R. capsulata DNA from Escherichia coli to a set of R. capsulata mutants. Plasmids pDPT51 and pDPT55 were constructed to mediate the intergeneric mobilization of pBR322 derivatives, and a mutant of R. capsulata with improved intergeneric recipient activity was isolated. Four previously unmapped genes affecting bacteriochlorophyll synthesis and two genes affecting photochemical reaction center synthesis have been located by marker rescue. Some of the fragments of R. capsulata DNA are capable of vector-independent complementation, implying that promoters are located on these fragments. Other fragments complement only in one orientation of insertion in the vector, implying transcription from promotors on the vectors and thereby fixing the direction of transcription for those fragments. Still other fragments of DNA show rescue only via recombination between homologous plasmid-borne DNA fragments and chromosomal mutations. The physical dimensions of the genetic map are 3.0 megadaltons per map unit, which agrees with previous estimates based on the size of the R. capsulata gene transfer agent.

Abstract

We report here the sequence of a 375-bp EcoRI-AvaI DNA fragment that previously has been shown to contain a locus (termed partition or par) responsible for stable maintenance of the pSC101 plasmid in growing cell populations. The DNA sequence of the par region encodes no obvious proteins and contains no segments having the structural characteristics of transcriptional or translational start signals. However, segments of the par locus appear capable of forming regions of intra-strand secondary structure, one of which resembles a rho-independent transcription terminator. Computer analysis shows regions within par that have homology with sequences found near the origin of replication of the Escherichia coli chromosome and of the pBR322 and ColE1 plasmids. The par sequence homology in the pBR322 and ColE1 plasmids maps in the vicinity of sites that interact with the E. coli replication factor Y and accomplishes initiation of DNA synthesis on single-strand templates.

Abstract

The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed.

DECAY OF MESSENGER-RNA IN ESCHERICHIA-COLI - INVESTIGATION OF THE FATE OF SPECIFIC SEGMENTS OF TRANSCRIPTSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESVONGABAIN, A., Belasco, J. G., Schottel, J. L., Chang, A. C., Cohen, S. N.1983; 80 (3): 653-657

Abstract

An assay was developed to investigate the fate of specific segments of beta-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an endonuclease capable of cleaving single-stranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNA . DNA hybrids were subjected to S1 nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini.

Abstract

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.

Abstract

The amino-terminal sequence of the Tn3 transposase protein was determined to be Pro-Val-Asp-Phe-Leu-Thr-Thr-Glu-Gln-Val-Glu-Ser.... This was determined both from an active transposase protein purified from a transposase overproducing mutant strain and from a hybrid transposase-beta-galactosidase fusion protein. The amino acid sequence corresponded to the DNA sequence of the transposase gene beginning at an ATG initiation codon, as previously predicted from the analysis of transposase-beta-galactosidase gene fusions.

Abstract

Five single base pair mutations that increase expression of the tnpA (transposase) gene of the Tn3 transposon approximately 30-fold, but which still allow the gene to be regulated, have been isolated by using a generally applicable procedure that involves distally linked lac gene fusions. The mutations, which are all located in a region controlling initiation of translation of the tnpA gene, do not affect normal repression of tnpA by the tnpR gene product, and yield up to a 9000-fold increase in tnpA protein production when combined with a tnpR mutation and placed on a high copy number plasmid. The mutation yielding the highest expression level was separated from the fused lac gene segment by homologous recombination and was found to increase the rate of transposition without altering the nature of the transposition product; in cells defective in both the E. coli recA gene and the tnpR gene of tn3, cointegrate transposition-intermediate structures occur with the overproducing--as well as with the wild-type--tnpA gene. In the presence of a functional Tn3 tnpR gene or the related transposon delta gamma, such cointegrate structures are resolved into the final products of transposition.

Abstract

Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.

Abstract

When strains of Streptomyces coelicolor A3(2) lacking the previously identified autonomous plasmids SCP1 and SCP2 are crossed with Streptomyces lividans 66, some of the S. lividans progeny are able to elicit zones of growth inhibition (lethal zygosis), previously associated with the transfer of conjugative Streptomyces plasmids, when grown in contact with S. lividans 66. Some such progeny yield covalently closed circular (CCC) plasmid DNA, the size and restriction endonuclease cleavage pattern of which is constant for a particular isolate, but varies among isolates. These plasmids, which have been named SLP1.1, SLP1.2, etc., all confer resistance to lethal zygosis elicited by the others. Genetic and molecular characterization of the plasmids reveals that they are derived from the strA region of the chromosome of S. coelicolor. It is proposed that, before or during mating with S. lividans, the SLP1 sequences are excised from the chromosome, bringing varying regions of the surrounding chromosome with them, and can circularize to yield the SLP1 family of plasmids. Autonomous SLP1 plasmids can also be generate by cleaving total DNA of S. coelicolor with certain restriction enzymes, ligating it, and transforming the DNA into S. lividans. The autonomous SLP1 plasmids exist within S. lividans in a few copies per chromosome, and act as fertility factors. They provide suitable vectors for DNA cloning since the segments of chromosomal DNA carried by the larger members of the family are dispensable.

CLONING AND ANALYSIS OF STRONG PROMOTERS IS MADE POSSIBLE BY THE DOWNSTREAM PLACEMENT OF A RNA TERMINATION SIGNALPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESGentz, R., Langner, A., Chang, A. C., Cohen, S. N., Bujard, H.1981; 78 (8): 4936-4940

Abstract

Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo.

Abstract

We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.

Abstract

Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment.

Abstract

Cordycepin-5'-triphosphate (3'-deoxyadenosine-5'-triphosphate) can be incorporated into the 3'-ends of DNA fragments using terminal deoxynucleotidyl transferase from calf thymus (Bollum, 1974). Because cordycepin-5'-monophosphate lacks a 3'-OH group, only a single residue is incorporated. Furthermore, DNA molecules that contain cordycepin-5'-monophosphate at their 3'-ends become resistant to hydrolysis by exonucleases that require free 3'-OH ends. As an alternative to 5'-end labeling of complementary DNA strands, we have used [32P]cordycepin-5'-triphosphate labeling of 3'-ends to confirm the nucleotide sequence of a HhaI-endonuclease-generated pTU4-plasmid DNA fragment that contains several hot spots for insertions of the transposable genetic element Tn3. 3'-End labeling with [32P] cordycepin-5'-triphosphate has also proved useful in determining the sequence of the pTU4 DNA in the vicinity of a strategically located SstII endonuclease cleavage site in the replication region of the plasmid.

Abstract

247 independent events involving insertion of the TN3 transposable element into a 4 kb constructed plasmid (pTU4) of partially known DNA sequence were studied by restriction endonuclease mapping, and 65 of these insertion sites were examined further by DNA sequence analysis. Our results show that the previously proposed regional specificity for Tn3 insertion is associated with a strong preference for AT-rich segments as insertion sites. Moreover, multiple insertions of the Tn3 occurred at certain AT-rich nucleotide positions, and 23 of 26 independent insertion events at a single nucleotide position were found to be in the same orientation. A region of the recipient plasmid showing major homology with the terminal 18 bp of Tn3 was identified in the vicinity of an 11 nucleotide segment that included three insertional hot spots and 36 independent insertions. Our results indicate that the site and orientation of insertion of Tn3 are at least partly determined by the primary nucleotide sequence of the recipient genome, and suggest that insertional hot spots may result from the combined effects of AT richness plus homology of the recipient genome with the terminal sequences of Tn3.

STRUCTURAL ORGANIZATION OF HUMAN GENOMIC DNA ENCODING THE PRO-OPIOMELANOCORTIN PEPTIDEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESChang, A. C., Cochet, M., Cohen, S. N.1980; 77 (8): 4890-4894

Abstract

We have isolated a human genomic DNA segment encoding the corticotropin-beta-lipotropin precursor peptide from a fetal DNA library, using previously cloned bovine cDNA for this peptide as a probe. The human genomic DNA was studied by electron microscope heteroduplex analysis and gel blotting methods, and its nucleotide sequence was determined and compared with that of cDNA corresponding to bovine pro-opiomelanocortin mRNA. From this sequence, segments of interspecies conservation and divergence, punctuated by pairs of the basic amino acid residues lysine and arginine, were identified. No noncoding intervening sequence was observed over an 830-base-pair DNA segment extending from a position near the 5' end of the structural pro-opiomelanocortin gene through the 3' terminus of the cDNA and including sequences for the component peptide hormones corticotropin and beta-lipotropin.

Abstract

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5' untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.

Abstract

We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.

Abstract

Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.

Abstract

We have identified and characterized a genetic function (designated par, for partition) that is required for stable maintenance of plasmids within exponentially growing cell populations. This function, which accomplishes the active distribution of plasmid DNA molecules to daughter cells, has been localized within the pSC101 plasmid to a 270 bp segment adjacent to the replication origin. The par locus, which appears to be functionally equivalent to the centromere of eucaryotic cells, is able to rescue unstable pSC101-derived replicons or an unrelated par- P15A-derived multicopy replicon in the cis, but not the trans, configuration. It is independent of copy number control and dose not specify plasmid incompatibility. Furthermore, it is not associated directly with plasmid replication functions.

Abstract

Insertion of Tn3 generates a five base pair repeat of a nucleotide sequence indigenous to the recipient genome. Tn3 promoted deletions extend precisely from the Tn3 terminus and remove one of the 5 base pair repeats while not affecting the ability of Tn3 to subsequently undergo translocation. A direct repeat of a 10 bp sequence located in the Tn3 termini occurs internally within Tn3 and may affect the orientation of insertion.

LACTOSE GENES FUSED TO EXOGENOUS PROMOTERS IN ONE-STEP USING A MU-LAC BACTERIOPHAGE - INVIVO PROBE FOR TRANSCRIPTIONAL CONTROL SEQUENCESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACASADABAN, M. J., Cohen, S. N.1979; 76 (9): 4530-4533

Abstract

The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage. This phage also carries a gene encoding resistance to ampicillin (Ap)[Mu(Ap, lac)]. After infection and upon establishment of lysogeny, the Mu(Ap, lac) genome can integrate into apparently random sites in the Escherichia coli chromosome. When integration occurs within a gene in the orientation of its transcription, the lactose structural genes are so situated that they become expressed solely from the promoter of that gene. Thus, expression of the lactose genes of Mu(Ap, lac) can be used as an assay for transcription of that gene and for functional and mutational studies of gene regulation.

Abstract

The effect of temperature on the translocation frequency of the Tn3 element was investigated. The temperature optimum for translocation of Tn3 was in the range from 26 to 30 degrees C. At temperatures above 30 degrees C, the translocation frequency decreased rapidly and linearly; at 36 degrees C it was only 5% of the frequency observed at 30 degrees C. The duration and reversibility of the temperature effect were utilized to demonstrate a requirement for protein synthesis in the translocation process.

STRUCTURAL AND FUNCTIONAL-ANALYSIS OF CLONED DNA SEGMENTS CONTAINING THE REPLICATION AND INCOMPATIBILITY REGIONS OF A MINI-PLASMID DERIVED FROM A COPY NUMBER MUTANT OF NR1JOURNAL OF BACTERIOLOGYTaylor, D. P., Cohen, S. N.1979; 137 (1): 92-104

Abstract

A 1.45-megadalton segment of DNA cloned from a miniplasmid derived in vivo from a copy number mutant of the R plasmid NR1 has been shown to contain all functions essential for incompatibility and autonomous plasmid replication in Escherichia coli. Specific endonuclease cleavage sites within this DNA segment that localize functions required for replication have been mapped. A 0.45-megadalton fragment that specifies the FII incompatibility of NR1 has been identified within the replication region, and DNA fragments containing this incompatibility region, but lacking other functions required for replication, have been cloned.

Abstract

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.

Abstract

A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycol-induced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4 x 10(7) transformants per microgram of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.

Abstract

An online computer-based system for monitoring patients for potential adverse drug reactions during their hospital stay is described. The adverse drug reaction monitoring system uses a Digital Equipment Corporation (DEC) PDP-11 computer which is programmed in MUMPS (Masschusetts General Hospital Utility Multi-programming System). Primary references from the medical and scientific literature are analyzed and evaluated before being included in the data base. The patient's adverse reaction history, obtained by nursing personnel during a patient interview, is entered into the computer by pharmacy staff. The computer screens new prescription orders for potential adverse reactions; any adverse reaction reports are sent to the patient's physician. Other special programs that are used in the patient monitoring system include medication profiles, drug-drug interaction screening and prescription discontinuation date entry. The computer system enable detection of potential adverse drug reaction and notification of the patient's physician prior to administration of the prescribed drug.

IDENTIFICATION AND CHARACTERIZATION OF A SELF-REGULATED REPRESSOR OF TRANSLOCATION OF THE TN3 ELEMENTPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChou, J., CASADABAN, M. J., Lemaux, P. G., Cohen, S. N.1979; 76 (8): 4020-4024

Abstract

Gene fusions that bring expression of the lacZ gene under control of transcriptional and trnaslational signals within the transposable element Tn3 have been used to study regulation of Tn3-specified proteins. A gene encoding a 21,355-Mr peptide that represses translocation of Tn3 and acts at the level oquenced; amber, missense, and cis-dominant (operator-constitutive) point mutations in this gene have been isolated and characterized.

Abstract

Fusions that bring lac gene expression under the control of transcriptional and translational signals within the Tn3 element have been used to identify and characterise a Tn3-encoded 'transposase' (Tnp) peptide of MW 100,000 essential for transposition. The gene specifying this product is regulated by the Tn3 repressor protein and is part of a bidirectional genetic unit that includes the repressor gene.

Abstract

The performance of a computer-based clinical consultation system is evaluated. The program, called MYCIN, is designed to function as an aid for infectious disease diagnosis and therapy selection, with an initial emphasis on bacteremias. The evaluation methodology is discussed, as well as the difficulties encountered in attempting to evaluate clinical judgments. Specialists in infectious diseases judged MYCIN's final therapy recommedation, and intermediate conclusions about the significance of the infection and identity of infecting organisms. The evaluation techniques described may be useful in assessing the performance of other clinical decision aids. Results of the evaluation show that the program's therapy recommedations meet Stanford experts' standards of acceptable practice 90.9% of the time (table 2), with some variation noted both among individual experts and between Stanford experts and others (tables 1, 2).

Abstract

An evaluation of a computer-based consultation system called MYCIN was made. Eight independent evaluators with special expertise in the management of meningitis compared MYCIN's choice of antimicrobials with the choices of nine human prescribers for ten test cases of meningitis. MYCIN received an acceptability rating of 65% by the evaluators; the corresponding ratings for acceptability of the regimen prescribed by the five faculty specialists ranged from 42.5% to 62.5%. The system never failed to cover a treatable pathogen while demonstrating efficiency in minimizing the number of antimicrobials prescribed. The study design may be useful in assessing the performance of other computer-based clinical decision-making systems.

Abstract

The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1. The inability of the composite plasmid to replicate at 42 degrees in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nervertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.

Abstract

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.

Abstract

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

Abstract

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.

PHENOTYPICALLY CRYPTIC ECORI ENDONUCLEASE ACTIVITY SPECIFIED BY COLE1 PLASMIDPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMiller, C. A., Cohen, S. N.1978; 75 (3): 1265-1269

Abstract

An endonuclease having EcoRI specificity is produced by bacteria containing the ColE1 plasmid. Such bacterial cells fail to express restriction or modification functions in vivo, and phage or plasmid DNA obtained from ColE1-containing cells has unmodified EcoRI sites that are cleaved in vitro by purified EcoRI endonuclease or by enzyme extracted from bacteria that carry ColE1. No EcoRI DNA methylase activity associated with ColE1 has been detected. The finding of phenotypically cryptic ColE1-dependent EcoRI endonuclease activity and the absence of any detectable EcoRI modification system in ColE1-containing cells suggest a control mechanism that appears to prevent functional expression of the ColE1-determined enzyme in vivo.

Abstract

DNA fragments generated by the EcoRI of HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColE1 or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants. Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable supporting replication of a linked ColE1 plasmid in polA- bacteria, were also identified.

Abstract

mRNA that encodes the common peptide precursor for the hormones corticotropin and beta-lipotropin was purified from the neurointermediate lobe of bovine pituitaries, and double-stranded cDNA species synthesized from this template were cloned in Escherichia coli X1776 by inserting them into the Pst I endonuclease cleavage site of the pBR322 plasmid using poly(dG)poly(dC) homopolymeric extensions. Certain of the cloned cDNA inserts contain nucleotides corresponding to the complete amino acid sequence of bovine corticotropin and a coding sequence that corresponds to at least the first portion of bovine beta-lipotropin. The nucleotide sequences coding for corticotropin and beta-lipotropin are separated on the cDNA by a 6-base-pair sequence encoding lysine and arginine, indicating that the carboxyl terminus of corticotropin is connected on the precursor peptide with the amino terminus of beta-lipotropin by these two amino acids. In addition, the cloned cDNA insert is characterized by an unusually high C+G nucleotide base content as well as by a number of DNA sequence duplications.

Abstract

A procedure is described that selects for the insertion of transposable antibiotic resistance elements in a variety of recipient replicons. The selected translocation procedure, which employs a plasmid having a temperature-sensitive defect in replication as a donor of transposable genetic elements, was used to investigate certain characteristics of the translocation process. Our results indicate that translocation of the Tn3 element from plasmid to plasmid occurs at a 10(3)- to 10(4)-times-higher frequency than from plasmid to chromosome. In both cases, continued accumulation of Tn3 on recipient genomes is prevented by development of an apparent equilibrium when only a small fraction of molecules in the recipient population contain Tn3. An alternative method for estimation of translocation frequency has shown that the translocation process is temperature sensitive and that its frequency is unaffected by the presence of host recA mutation. Insertions of Tn3 onto the 65 X 10(6)-dalton R6-5 plasmid in Escherichia coli are clustered on EcoRI fragments 3 (8 of 23 insertions) and 9 (7 of 23 insertions), which contain 12 and 5%, respectively, of the R6-5 genome. The occurrence of multiple insertions of Tn3 within EcoRI fragment 9, which contains the IS1 element and a terminus of the Tn4 element, is consistent with earlier evidence indicating that terminal deoxyribonucleic acid sequences of already present transposable elements may provide recognition sequences for subsequent illegitimate recombinational events.

INVIVO SITE-SPECIFIC GENETIC-RECOMBINATION PROMOTED BY ECORI RESTRICTION ENDONUCLEASEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChang, S., Cohen, S. N.1977; 74 (11): 4811-4815

Abstract

Site-specific genetic recombinations promoted in vivo by the EcoRI endonuclease has been demonstrated by using constructed hybrid plasmids in which the chloramphenicol resistance gene was inactivated by insertion of DNA fragments at an EcoRI site within the gene. Such recombination can involve either the joining of intracellularly generated cohesive termini of the same DNA fragment or intermolecular ligation of different DNA fragments. DNA cleavage and ligation in vivo are precise: recombinant DNA molecules show functional continuity of the gene sequence cleaved by the enzyme and regeneration of nucleotide recognition sites for both the EcoRI endonuclease and the EcoRI DNA methylase. In other experiments, EcoRI-generated fragments of eukaryotic DNA that had not been modified by the Escherichia coli K methylase were shown to be taken up by bacterial cells and to undergo intracellular ligation to segments of bacterial plasmid DNA.

Abstract

Physicians at Stanford University Medical Center were surveyed to evaluate their responses to a computerized drug interaction warning system one year after its installation. While one-fourth of the 862 respondents had personally received at least one warning report, the educational benefits of the system had diffused to many physicians in the hospital. Reports were found to be useful for patient management and 44 per cent of the physicians who had received reports indicated they had changed their behavior in response to the information. The system was particularly beneficial for interns and medical students, and appears to have potential as an educational tool for training in therapeutics. While several problems with the system were noted, the overall attitude of the respondents toward it was favorable. The drug interaction warning system appears to have a promising future for reducing the problem of drug interactions among hospitalized patients.

Abstract

Although it carries two competent replication systems, a composite plasmid formed in vitro by linkage of the complete ColE1 and pSC101 plasmid replicons at their unique EcoRI endonuclease cleavage sites normally uses only the replication origin and functions of the ColE1 component. Restriction of ColE1 replication functions by DNA polymerase I deprivation results, however, in exclusive use of the pSC101 replication origin. When using the ColE1 replication system the composite plasmid is nevertheless incompatible with both the parent replicons. This suggests that a trans-dominant gene product is involved in plasmid incompatibility and supports negative control rather than positive control models for regulation of the initiation of DNA replication.

Abstract

Recent evidence suggests that plasmids have evolved by site-specific recombinational events involving translocation and insertion of discretely defined DNA segments. The role of translocating genetic elements and repeated DNA sequences in the formation and structural evolution of bacterial plasmids, and in the control of plasmid gene expression, is the subject of this brief review. Insertion sequence (IS) regions are discrete segments of DNA that are known to cause strongly polar mutations in the genes of Escherichia coli and several bacteriophages as a consequence of their insertion into bacterial or phage genomes. Recent investigations have identified three separate kinds of IS segments on plasmids, and have indicated that such regions may have a role in 1) site-specific reversible dissociation of antibiotic resistance plasmids into their component segments, 2) recombination of certain plasmids with the bacterial chromosome, and 3) translocation of segments of plasmid DNA onto other replicons, or onto different sites of the same replicon. In addition, such DNA sequences, which may be repeated on plasmid genomes in either direct or reverse orientation, are involved in the control of plasmid gene expression. Inverted repeats other than the genetically characterized IS segments also appear to be involved in recA-independent, recombination and translocation of plasmid DNA segments. These inverted repeats contain palindromic nucleotide sequences on each strand of DNA and are detectable as hairpin-loop structures by electron microscope heteroduplex analysis. Such palindromes resemble the recognition sites for restriction endonucleases, some of which are encoded by plasmids, suggesting that similar endonucleolytic enzymes may be involved in the translocation of plasmid DNA segments.

Abstract

Mycin, a computer-based consultation system which provides to physicians antimicrobial therapy recommendations for patients with bacterial infections, is described. The consultation program arrives at therapeutic decisions using a built-in knowledge base as well as patient data entered by the physician. The system is capable of explaining its recommendations and answering questions about its reasoning process. The system's knowledge can be updated and corrected easily by infectious disease experts. At present the system is operational within a research setting; its routine use in a clinical setting will require further evaluation of its reliability and effectiveness.

Abstract

Transposable elements of DNA that are structurally defined and genetically discrete units seem to have an important role in the evolution of bacterial plasmids. Recombination occurring at the termini of such elements can result in the joining together of unrelated DNA segments that lack extensive nucleotide sequence homology. In addition, transposable elements serve as novel biological switches capable of turning on and off the expression of nearby genes as a consequence of their insertion into or excision from plasmid genomes.

Abstract

Sucrose gradient analysis of total sea urchin DNA cleaved with the EcoRI and Hind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA with EcoRI produces 2.2 and 4.8 kb fragments, and which are contained in a 7 kb Hind III fragment. Cleavage with both enzymes reveals that the 2.2 kb EcoRI fragment contains a Hind III site 0.15--0.2 kb from an end. RNA.DNA hybridization between chimeric palsmic DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.

SITE-SPECIFIC RECA-INDEPENDENT RECOMBINATION BETWEEN BACTERIAL PLASMID - INVOLVEMENT OF PALINDROMES AT RECOMBINATIONAL LOCIPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKopecko, D. J., Cohen, S. N.1975; 72 (4): 1373-1377

Abstract

A recA-independent recombinational event is described which results in insertion of an entire plasmid genome at a unique site of another plasmid, and coincident excision of a precisely defined DNA segment originally present at the point of the insertion. The resulting recombinant molecules subsequently can undergo site-specific translocation of their component segments or inversion of their original DNA sequence orientation. The events observed entail nonreciprocal exchange of genetic material, and involve a discrete nucleotide sequences that is duplicated in rotationally symmetrical reverse orientation on plasmid DNA (i.e., inverted repeat; palindrome).

Abstract

An online computer-based system to monitor prospectively for potential drug interactions in a hospital setting is described. The system, developed at Stanford University Medical Center, is fully operational and is used to inform pharmacists, nurses and physicians as to the severity and speed of onset of potential drug interactions. In addition, the system can produce prescription labels and patient-drug profiles for the pharmacy and serves as a retrieval source of drug interaction information. Each report provides information regarnding the pharmacological effect and mechanism of the interactions, as well as statements involving relevant clinical findings associated with these interactions.

CLONING, ISOLATION, AND CHARACTERIZATION OF REPLICATION REGIONS OF COMPLEX PLASMID GENOMESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICATimmis, K., Cabello, F., Cohen, S. N.1975; 72 (6): 2242-2246

Abstract

EcoRI endonuclease-generated DNA fragments carrying replication regions of the F'lac and R6-5 plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6-5 replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions and the locus governing plasmid incompatibility has been shown for both parent replicons. The cloning method reported appears to be generally applicable for the identification and isolation of replication regions of a variety of complex genomes.

Abstract

Previous studies have shown that there is a deoxyribonucleic acid (DNA) segment, of length 1.3 kb and denoted as the alphabeta sequence, which occurs twice on the F plasmid at corrdinates 93.2 to 94.5/OF kb and 13.7 to 15.0F kb. In the present investigation, heteroduplexes were prepared between a phage DNA carrying the insertion sequence IS3 and suitable F-prime DNAs. The hybrids formed show that IS3 is the same as alphabeta. This result plus previous studies support the view that: (i) the insertion sequence IS2 and IS3 occur on F and, in multiple copies, on the main bacterial chromosome of Escherichia coli K-12; and (ii)these IS sequences on the main bacterial chromosomes are hot spots for Hfr formation by reciprocal recombination with the corresponding sequences of F.

Abstract

Histone genes have been cloned selectively in Escherichia coli directly from unfractionated sea urchin DNA using labelled mRNA probe. The cloning method used is potentially applicable for isolating any genetic sequence for which a probe is available. The several eukaryotic gene fragments introduced into bacteria by this method collectively contain all or nearly all of the DNA sequences represented in histone mRNA.

Abstract

A procedure is described that uses an indicator plasmid (pSC201) to identify cells in a bacterial population that have been co-transformed with a second plasmid lacking detectable phenotypic properties. Under appropriate conditions of indirect selection, between 50 and 85% of transformants carrying the indicator plasmid also contain the nonselected plasmid. A temperature-sensitive mutation in the replication functions of the indicator plasmid enables its elimination from doubly transformed bacteria. Using this procedure, we have isolated bacteria that carry only the small cryptic plasmid. P15A, of the Escherichia coli strain 15. This genetic element, which contains only 2,300 nucleotide pairs, is thus capable of functioning as a replicon independently of the two larger plasmids normally associated with it in E. coli 15 strains (Ikeda, Inuzuka, and Tomizawa, 1970).

Abstract

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.

Abstract

The mouse mitochondrial DNA genome has been cloned in Escherichia coli by linking it to the pSC101 plasmid replicon at cohesive-ended cleavage sites generated by Eco Rl restriction endonuclease. The four possible configurations of chimeric molecules that contain the nucleotide sequences of mitochondrial DNA in their native relationship were distinguished by Hind III restriction endonuclease digestion and electron microscopic heteroduplex analysis. Chimeric molecules utilize the pSC101 replication origin and do not maintain the "D-loop" region or the low frequency of ribonucleotides found in native mitochondrial DNA. Hybridization of the RNA synthesized in E. coli minicells carrying the four types of chimeras indicates that transcription occurs predominately on the light strand of the mitochondrial DNA in all cases. This result implies that initiation of RNA synthesis occurs within the mitochondrial DNA segment. Although specific polypeptide synthetis is directed by the mitochondrial DNA segment of each of the chimeras in E. coli minicells, the molecular weight distribution of the polypeptides synthesized consists primarily of low molecular weight species and thus differs from that observed in mitochondria in mouse L cells.

UTILIZATION OF 2 DISTINCT MODES OF REPLICATION BY A HYBRID PLASMID CONSTRUCTED INVITRO FROM SEPARATE REPLICONSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICATimmis, K., Cabello, F., Cohen, S. N.1974; 71 (11): 4556-4560

Abstract

A hybrid plasmid, pSC134, that codes for two distinct sets of replication functions has been constructed in vitro by ligation of EcoRI endonuclease-cleaved pSC101 and Col E1 plasmid replicons, and has been introduced into Escherichia coli by transformation. The replication properties of the pSC134 plasmid in DNA polymerase I-defective mutants or in the presence of chloramphenicol indicate that this hybrid plasmid can utilize the functionally distinct modes of replication specified by both of its parent replicons.

REPLICATION AND TRANSCRIPTION OF EUKARYOTIC DNA IN ESCHERICHIA-COLIPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMorrow, J. F., Cohen, S. N., Chang, A. C., BOYER, H. W., Goodman, H. M., Helling, R. B.1974; 71 (5): 1743-1747

Abstract

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA.

GENOME CONSTRUCTION BETWEEN BACTERIAL SPECIES INVITRO - REPLICATION AND EXPRESSION OF STAPHYLOCOCCUS PLASMID GENES IN ESCHERICHIA-COLIPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChang, A. C., Cohen, S. N.1974; 71 (4): 1030-1034

Abstract

Genes carried by EcoRI endonuclease-generated fragments of Staphylococcus plasmid DNA have been covalently joined to the E. coli antibiotic-resistance plasmid pSC101, and the resulting hybrid molecules have been introduced into E. coli by transformation. The newly constructed plasmids replicate as biologically functional units in E. coli, and express genetic information carried by both of the parent DNA molecules. In addition, electron microscope heteroduplex analysis of the recombinant plasmids indicate that they contain DNA sequences derived from E. coli and Staphylococcus aureus. Recombinant molecules can transform other E. coli cells for penicillin-resistance markers originally carried by the staphylococcal plasmid, and can be transferred among E. coli strains by conjugally proficient transfer plasmids.

Abstract

A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.

RECIRCULARIZATION AND AUTONOMOUS REPLICATION OF A SHEARED R-FACTOR DNA SEGMENT IN ESCHERICHIA-COLI TRANSFORMANTS - (PLASMID-TRANSFORMATION-ANTIBIOTIC RESISTANCE DNA)PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N., Chang, A. C.1973; 70 (5): 1293-1297

Abstract

Controlled shearing of R-factor DNA leads to formation of fragments carrying an antibiotic resistance gene present on, but not expressed by, the intact R-factor. Transformation of CaCl(2)-treated E. coli by such fragments yields an autonomously replicating tetracycline-resistance plasmid (Tc6-5) that contains only a small fraction of the genome of the parent R-factor, and lacks both its fertility functions and its other drug-resistance determinants. Although the Tc6-5 plasmid is not self-transmissible, it can interact and/or recombine with conjugally-proficient plasmids that promote its transfer to other bacteria.

CONSTRUCTION OF BIOLOGICALLY FUNCTIONAL BACTERIAL PLASMIDS IN-VITROPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N., Chang, A. C., BOYER, H. W., Helling, R. B.1973; 70 (11): 3240-3244

Abstract

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.

Abstract

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.

NONCHROMOSOMAL ANTIBIOTIC RESISTANCE IN BACTERIA - GENETIC TRANSFORMATION OF ESCHERICHIA-COLI BY R-FACTOR DNAPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N., Chang, A. C., Hsu, L.1972; 69 (8): 2110-?

Abstract

Transformation of E. coli cells treated with CaCl(2) to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance, fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R-factor DNA in this system.

NON-CHROMOSOMAL ANTIBIOTIC RESISTANCE IN BACTERIA .3. ISOLATION OF DISCRETE TRANSFER UNIT OF R-FACTOR-R1PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N., Miller, C. A.1970; 67 (2): 510-?

Abstract

A covalently-closed circular DNA species, banding at a buoyant density of rho = 1.709 g/cm(3) in CsCl, has been identified in antibiotic-sensitive colonies of E. coli strain AB2463 (rec A(-)) after mating with a Proteus mirabilis strain that carries the R-factor, R1. This plasmid, which represents a stable segregant of R1 that has lost all of the drug resistance determinants present on the parent R-factor but which has retained its ability to be transferred by conjugation, fulfills the functional definition of the R-factor transfer unit (RTF).

TRANSCRIPTION OF COMPLEMENTARY STRANDS OF PHAGE LAMBDADNA IN VIVO AND IN VITROPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACohen, S. N., Hurwitz, J.1967; 57 (6): 1759-?